Dry powder formulation from fruits of Physalis peruviana L. standardized
extract with hypoglycemic activity

Carlos-A. Bernal, Marcela Aragón, Yolima Baena

PII: S0032-5910(16)30400-4
DOI: doi: 10.1016/j.powtec.2016.07.008
Reference: PTEC 11770

To appear in: Powder Technology

Received date: 9 March 2016

Revised date: 9 June 2016
Accepted date: 3 July 2016

Please cite this article as: Carlos-A. Bernal, Marcela Aragón, Yolima Baena, Dry powder
formulation from fruits of Physalis peruviana L. standardized extract with hypoglycemic
activity, Powder Technology (2016), doi: 10.1016/j.powtec.2016.07.008

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 ACCEPTED MANUSCRIPT 1

Dry powder formulation from fruits of Physalis peruviana L. standardized extract with

hypoglycemic activity

Authors

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Carlos-A. Bernala, Marcela Aragóna, Yolima Baenaa,*

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a
Universidad Nacional de Colombia – Sede Bogotá – Science Faculty –Pharmacy Department– Research Group

Technology of Natural Products – Carrera 30 # 45-03, , Bogotá D.C., (111311), Colombia.

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Abstract
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The aims of this study were to develop a dry powder formulation from standardized extract of fruits of Physalis peruviana
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L., for oral administration, and to choose the best drying process for retaining the hypoglycemic activity, applying a

factorial type experimental design. The development of a pharmaceutical formulation from an herbal extract has
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technological complications arising from inappropriate pharmaceutical characteristics related to its extract nature as a
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multicomponent system. Physalis peruviana extract showed the following technology difficulties: high hygroscopicity

(which affects its flow, blend uniformity and stability), reduced flow properties (poor, non-uniform flow), high cohesivity,
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and low compressibility. To solve these problems, physical state changes of the extract (e.g. by absorption processes in a

substrate) could contribute to improving its physical properties. The selection of excipients for use in the formulation was

based on a compatibility study with binary solid mixtures (extract and each excipient) analyzed trough HPLC, and

additionally, applying a factorial statistical experimental design to choose the best absorbent for the extract and the best

drying method, evaluating like response variables: particle size, bulk and tap densities, angle of repose and

hygroscopicity. Finally, a hypoglycemic assay was conducted in mice, using the best formulation. According to the

compatibility study, the following excipients may be considered promising for use in a possible solid formulation from the

ethanolic extract of fruits of Physalis peruviana: Disintegrants, absorbents and diluents: microcrystalline cellulose and

corn starch; binder: polyvinylpyrrolidone. From the extract and the excipients selected as absorbents and binder

(microcrystalline cellulose PH 101, 102, 200, corn starch and polyvinylpyrrolidone), different granules were produced,

*
Corresponding author. Tel.: +57 1316 5000 ext. 14620 E-mail address: ybaenaa@unal.edu.co
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which were dried by two methods. The factorial experimental design showed that microcrystalline cellulose PH 102 and

corn starch were the best absorbents, and that drying in a fluidized bed was the best method, offering advantages in terms

of physicochemical properties such as particle size, density, voluminosity, flowability and hygroscopicity, compared to

the initial extract and to the other formulations. The hypoglycemic assay showed that the activity of the extract remained

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after its transformation to a solid state. The results suggest that the dry powder formulation obtained from the ethanolic

extract of fruits of Physalis peruviana may be of use as a future phytotherapeutic product, or a more stable intermediate

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herbal product than the extract, as an adjuvant in the treatment of diabetes.

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Keywords: Physalis peruviana L.; hypoglycemic activity; herbal product; pharmaceutics properties; statistical

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experimental design; compatibility study.

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1. Introduction
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In the last few years, the use of traditional medicine has increased. This therapeutic strategy is based on the application of
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phytomedicines on which active components are produced from drugs derived from plant material [1]. In 2007, total

European sales of pharmaceutical products were €176 billion [2]. The European Union (EU) pharmaceutical market has
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grown by approximately 4% since 2005. At the same time, sales of pharmaceutical products in East European countries,
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showed strong growth from 2005 to 2007, generated mainly by a significant increase in non-prescription sales.

Furthermore, total pharmaceutical production in the EU totaled approximately €182 billion in 2006, 35% of world’s
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production. According to the Development Center for Biotechnology, the global market value for herbal medicines was

€19 billion in 2006 [2]. This growth in the phytomedicines market has led to a considerable increase in attention focused

on the research and development of herbal products [3, 4].

Physalis peruviana is a plant of the solanaceae family, commonly known in Colombia as uchuva; usually, its fruits are

consumed as food and other parts of the plant are used for medicinal purposes [5, 6]. Native to the Andean Region of

South America, it grows in Bolivia, Chile, Colombia, Ecuador, Peru and Venezuela, reaching heights between 1700 and

2900 meters above sea level; the fruit is used for domestic consumption and for exportation [7]. The fruit is also used in

folk medicine for the prevention and treatment of pterygia, as an expectorant, diuretic and anthelmintic, for the treatment

of diabetes, albuminuria and pertussis, and to strengthen teeth and prevent tooth decay. According to users, daily

consumption of raw fruits of Physalis peruviana helps maintain stable blood glucose levels [8].
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Although knowledge and development methodologies for herbal products are similar to those required for conventional

drugs, due to their nature, herbal products require additional considerations [4, 9]. The formulation of an extract from

herbal material in a dosage form involves the efforts of different areas, such as pharmaceutics, phytochemistry, analytical

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chemistry, instrumental analysis and pharmacology. Unlike the pure active ingredients, the natural extracts contain small

but varying amounts of active ingredient(s) and large amounts of secondary compounds (organic and inorganic salts, acids

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and organic bases, saponins, polyphenols, tannins, sugars, polysaccharides, etc.), which together, have a significant

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influence on their appearance, creating the need for the extract to be adapted to a dosage form. In the selection of the most

adequate dosage form, different aspects must be evaluated, such as the solubility, stability and physicochemical properties

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of the extract, as well as the characteristics of its secondary compounds [4].

The development of a phytotherapeutic involves obtaining a product that is physically and chemically stable,
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technologically feasible, and biologically available. In most cases, the extract needs to be adapted to an oral solid dosage

form, as the starting point of this development [4, 9-12].

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Among the different dosage forms, the solid oral dosage forms are preferred, being the most used in modern medicine,

including natural medicine. Among the advantages of a solid dosage form by the oral route are that it allows
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administration by the patient, and offers convenience and simplicity. It also allows the administration of a precise dose,
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increasing stability. For these reasons, this dosage form is the main method of administration of bioactive substances to

achieve systemic effects, and used in 90% of phytotherapeutic products [4, 9].
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The aim of this study was to develop a dry powder formulation for oral administration, from ethanolic extract of fruits of

Physalis peruviana L., as a possible future herbal product or as an intermediate herbal product for the adjuvant treatment

of diabetes.

2. Materials and methods

2.1. Materials

Physalis peruviana fruits were collected in Subia, Cundinamarca, Colombia in January 2013. A voucher specimen of the

plant (574701) was identified and deposited at the Herbario Nacional Colombiano of the Universidad Nacional de

Colombia.
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Microcrystalline cellulose Avicel® PH 101, PH 102 and PH 200 (FMC Biopolymer, Philadelphia, PA, USA,

pharmaceutical grade), corn starch (Industrias del Maiz, Colombia, pharmaceutical grade), dibasic calcium phosphate

dihydrate Encompress® (JRS Pharma, Patterson, NY, USA, pharmaceutical grade), lactose monohydrate Pharmatose®

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100M (Unired Químicas S.A.S, Colombia, pharmaceutical grade) and polyvinylpyrrolidone PVP Kollidon® K30 (BASF,

Ludwigshafen, Germany, pharmaceutical grade) were used in the excipient compatibility study and in the selection of the

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absorbent and dry method study. Methanol (MeOH) (Fisher Chemical, Belgium, HPLC grade), acetonitrile (MeCN)

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(Tedia, USA, HPLC grade), and deionised water (W) (was obtained using a Merck Millipore Milli-Q water purification

equipment, Billerica, MA, USA), were used in the excipient compatibility study in the form in which they were received

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from the supplier. Ethanol (EtOH) 96% (supplied by Empresa de Licores de Cundinamarca-Colombia, Pharmaceutical

grade) was used for preparation of the fluid plant extract. Magnesium chloride, Calcium Chloride, Sodium Chloride and

Sodium sulfate were of analytical grade (Merck, Germany) and were used for the higroscopicity tests. Acarbosa ≥ 95%
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(Sigma-Aldrich, USA, analytical grade), propylene glycol USP (Dow Chemical Company, USA, pharmaceutical grade)

and glycerin USP (Disproalquímicos, Colombia, Pharmaceutical grade) were employed for the hypoglycemic assay.
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2.2. Preparation of the extract

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The entire fruits of Physalis peruviana were weighted accurately (5000 g), liquefied, dried (40°C) and triturated. The
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powder obtained was extracted with EtOH. The extraction was performed using the percolation method, with continuous

additions of solvent. The extraction stage was carried out for 4 days, with a drug:solvent ratio of 1:15. The extract was
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concentrated on a rotary evaporator (IKA® RV-10) until a fluid extract was obtained.

2.3. Excipient compatibility study

2.3.1. Preparation of the samples

This study was performed following the methodology developed by Kopelman et al. [13]. Different binary mixtures

between the extract and some excipients that could be used as absorbents, or that will be part of the future development of

the phytotherapeutic product, were analyzed. The excipients selected for this study were: absorbents, diluents and/or

disintegrants (microcrystalline cellulose, corn starch, dibasic calcium phosphate and lactose) and binders

(polyvinylpyrrolidone). For this purpose, binary mixtures of ethanolic extract of Physalis peruviana fruits (75% -300mg)
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with absorbents (17.5% - 70 mg), disintegrants (6% -24 mg), diluents (17.5% - 70 mg) and binders (1 % - 4 mg) were

prepared by weighing the dry extract of the fruits and the required amount of each excipient, in type I glass vials; these

percentages are based on the expected composition in 400 mg of product. The vials were protected from light with

aluminum foil covers. Finally, 20 mg of water was added (5%) to each sample and mixed for 1 minute using a glass

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stirrer, to facilitate interactions between the excipients and the extract. The samples were then heated to a temperature of

50°C for 15 days. The extract without excipients (at 50°C and 5°C), was analyzed as negative control, following the same

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procedure, to determine the real influence that the excipients might have in the study [13-16].

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2.3.2. Analytical HPLC method

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The analysis of the phytochemical profile of the extract was performed through the previously validated HPLC analytical

method [17], selecting the most representative signals in the chromatographic profile for the wavelengths of 210, 254, 280
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and 350 nm; more detail information can be found in supplementary content. The samples were diluted to 1 mg/ml of

extract in MeOH HPLC grade, and filtered through a membrane filter (0.45 μm, Millipore); 10 microliters of the resulting
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filtrate was injected into the HPLC system equipped with Agilent 1200 series with a UV-DAD detector, and a column RP-
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18 LiChroCART® (250-4). As mobile phase, MeCN:W was used in concentration gradient: 0-1 min. (95:5); 1-61 min.

(5:95); 61-71 min. (5:95); 71-75 min. (95:5); 75-86 min. (95:5) at a flow rate of 1 ml/min.
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2.3.3. Data analysis

For statistical analysis of the data, the area of the most representative signals of each profile at each wavelength were
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compared with the negative control, employing the f2 similarity test, using Eq. (1), as in similar previous studies [13]. In

the Eq (1), Rt and Tt are the percentages of the areas under the curve of the selected characteristic signals (t = signal) for

the reference (negative control) and test profiles (binary mixtures), respectively. When f2 = 50-100, the two profiles are

considered similar because the values included in this range imply a difference of 10% or less at each point of the

comparison.

(1)

2.4. Development of a dry powder formulation.

2.4.1. Selection of the absorbent and drying method

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To determine the best absorbent for the extract, and the best drying method, a statistical experimental design (SED) with

randomized block was used after the absorption and in some cases after the agglutination, as shown in table 1. For this

purpose, the corn starch and microcrystalline celluloses PH 101, 102 and 200 were evaluated as individual absorbents.

Additionally, two combinations of materials were included (in one of these, agglutination with PVP was performed),

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using a mixture of corn starch and microcrystalline cellulose PH 102, this excipient was chosen for the tests because it has

an intermediate behavior compared to the other two celluloses used, in terms of particle size, bulk density and tamped

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density [18]. Mixtures of corn starch and microcrystalline cellulose PH 102 were prepared in proportions of components

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of 87.5% for microcrystalline cellulose PH 102 and 12.5% for the starch, as recommended in the literature [18]. To the

mixtures that required agglutination, 1% PVP in ethanol was added, until agglutination point. The drying methods

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evaluated were static bed (oven trays Stokes, model 38B, USA) and fluidized bed (Glatt, model: Biolab, Germany).

****Table 1**** MA
These formulations were manufactured by placing the absorbent material into a planetary mixer (Mixer kitchen aid

classic, model K4555, USA). Next, the extract was added and the blend was mixed until the extract was fully
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incorporated.
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The formulation obtained was passed through a No.20 mesh (U.S. standard testing sieve, USA, meets ASTM E-11
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specifications) to break down clusters and homogenize the particle size. Next, the solid material was dried: in the first
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case, in a circulating air oven, where it was carefully spread on a tray and allowed to dry for 8 hours at 40°C, and in the

second situation, in a fluidized bed, after preheating for 20 minutes in a circulating air oven (to remove excess of alcohol).
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The formulation was subjected to heating for 90 minutes at the same temperature. The dry material obtained was passed

through a No.20 mesh, collected in a plastic bag, and weighed. The following response variables were evaluated:

2.4.2. Physical-mechanic properties

Size and particle size distribution

Particle size and distribution were determined through the sieving method described by USP 38 [19] and by Martin et al.

[20], standardized in previous works [12, 21, 22], using 50 g of the formulation at a constant vibration intensity per twenty

minutes. For this test, ten repetitions were performed.

To determine the particle size, different statistical diameters were calculated according to Eq (2), where "n" is the weight
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retained by the lower sieve, in grams, and “d” is the arithmetic mean of the aperture in micrometers [20]. The size

distribution was obtained with the frequency distribution curve [20].

(2)

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Bulk and tap densities

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To determine the density of the formulations, the method described by Rodriguez et al was used [12]. For this procedure 1

gram of material, precision weighed, was gently poured into a 10 mL graduated cylinder, leaving it to fall freely and

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measuring the volume occupied by the material to determinate the Bulk Density (BD). Next, the material was tamped by

applying vibration, and the volume occupied was measured to calculate the Tap Density (TD). The compressibility of the

material was assessed using Carr's Compressibility Index (CI) shown in Eq. (3). Ten repetitions were performed for this
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test.
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(3)
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Angle of repose
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The flow properties of the formulations were determined by the static method, based on the measure of the angle of

repose. For this determination, 10 g of material were weighed and poured through a funnel positioned at a fixed height
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above a piece of graph paper on a flat horizontal surface, on which the material was allowed to form a cone. The height

(h) and radius (r) of the cone were then measured. The tangent of the angle of repose is given by the expression h/r ratio

[12]. For this test, ten repetitions were made.

Hygroscopicity

Samples of 1 gram of the formulations were weighed on an analytical balance Ohaus Model PA 214, in triplicate, and

poured into vial bottles. The uncovered samples were placed in different humidity environments at 20°C and weighed at

intervals of 1, 2, 4, 8, 12, 24, 48, 96, 120, 144, 168 and 264 hours [23, 24] . The Relative Humidity (RH) environments

were prepared and controlled using saturated salt solutions of magnesium chloride (38% RH), Calcium Chloride (52%

RH), Sodium Chloride (72% RH) and Sodium sulfate (97% RH). The percentage of gain or loss in each of the
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environments defined for the study was calculated by determining the change in the weight of the formulations. These

changes were compared with the behavior of the dry extract.

2.4.2.1. Statistical evaluation of the experimental design

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The data from the results of all the variables corresponding to each formulation were evaluated statistically by ANOVA

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(Analysis of Variance), with a significance level of 0.05, using the statistical package "R" version 2.10.1. This test was

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performed to determine whether the results showed statistically significant differences between them, and to evaluate the

influence of the excipients and/or equipment used in the study. To determine whether there was statistically significant

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variation for these variables, the assumptions of normality (Shapiro-Wilk test) and homoscedasticity (Test F) were

assessed. The datasets that met the assumptions of normality and homoscedasticity were considered to compare means by

the student t test, with a significance level of 0.05; data that did not meet the assumptions were examined by the Wilcoxon
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test, with a significance level of 0.05.
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2.5. Hypoglycemic assay

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This assay was performed for the extract and after being subjected to the formulation in solid state, to establish whether
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excipients or technological changes could have some influence on its pharmacological activity, which had been previously
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demonstrated [8, 25].

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2.5.1. Animals

ICR mice of 30-35 g in weight and 10 weeks old were used. The mice were bred and housed under standard conditions

(12 h light/12 h dark cycles, temperature 22 ± 2°C). The study was approved by the Ethics Committee of the Faculty of

Science, Universidad Nacional de Colombia (Act 03, May 23, 2011).

2.5.2. Hyperglycemic induction by oral overload of starch

Fasted animals were divided in four groups and were administered orally according to table 2 (n= 10 animals per group).

Blood Glucose Levels (BGL) were measured at 0, 1, 2 and 3 hours after starch administration. Blood samples were taken

by puncture of the retroorbital plexus. To measure the BGL, blood samples were analyzed using test strips (Accu-Chek
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Advantage II Control mg / dL Roche®) in a glucose meter (Accu - Chek). At the end of the experiment all animals were

euthanized by cervical dislocation.

***Table 2****

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3. Results and discussion

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3.1. Preparation of extract

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The dried fruit extract of Physalis peruviana L. was of a dark brown color, with characteristic odor, acid orange flavor,

and a very bitter taste that stays in the mouth; its texture was very pasty and sticky, with poor viscosity, making it difficult

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to handle. Additionally, it was moderately hygroscopic. The ethanolic extract of the fruits of Physalis peruviana L.

showed oral hypoglycemic activity in mice [8, 26]. The chloroform fraction obtained by solvent fractionation from an
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ethanolic extract enhanced the effect of exogenous insulin in normoglycemic mice [8].
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3.2. Excipient compatibility study

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In the design and development of a pharmaceutical product, it is essential to conduct compatibility studies in the
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preformulation stage, in order to make a proper selection of excipients. The results shown in Fig. 1 demonstrate the
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complexity of phytochemical profiling, which makes it difficult to interpret the results of the compatibility data; therefore,

it is preferable to consider the influence of the excipients in all important phytochemical markers within the matrix,

instead of just one or two marker components. One approach for comparing profiles of several components is to use
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statistical similarity metrics [13].

****Figure 1****

These methods have been mentioned as direct indicators for comparing profiles or curves; since the shapes of the two

profiles are directly compared, all data points can be used. However, for this study, the most representative signals in the

chromatographic profiles obtained by HPLC were selected. The results of the similarity test for compatibility of excipients

are shown in table 3; the values of f2 were calculated with the area of the most representative signals of each profile from

each wavelength, and compared with the negative control ( Fig.1). If the value obtained is 50-100, the two profiles could

be considered similar, as mentioned in the methodology; in general, it seems that the excipients do not affect the

chromatographic profile. Although the excipients are considered pharmacologically inert, they could catalyze, propagate
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or participate in physical or chemical interactions with the drug or in this case, with the Physalis peruviana extract, which

could compromise its quality or pharmacological performance. Chemical interactions can cause degradation of the active

components of the extract, reducing the amount available to produce the desired therapeutic effect. Degradation of

products can also compromise the safety and/or tolerance of the drug contained in the extract. Furthermore, the physical

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interactions can affect the dissolution rate, dose uniformity, and ease of administration [27].

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****Table 3***

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According to the results, there are incompatibilities of the extract with dibasic calcium phosphate and lactose, while with

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the other excipients showed no apparent incompatibilities. Each particular case is explained below.

For the dibasic calcium phosphate at the wavelength of 280 nm, the value is lower than 50, so the profiles of the extract
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and this excipient cannot be considered similar, as explained above. The slightly alkaline nature of dibasic calcium

phosphate could contribute to degradation of many metabolites, considering that the ethanolic extract of Physalis
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peruviana has an acidic pH [13, 15, 28]. Although the dibasic calcium phosphate is a non-hygroscopic excipient [18],
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which makes it desirable for the physical stability of the formulated product, its chemical interaction with the extract

makes it unsuitable for use in a possible formulation.

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The results for the extract related to lactose show that the profiles were not similar at a wavelength of 330 nm, which

would evidence incompatibility. Lactose can participate in complex reactions with compounds with primary and
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secondary amines [18]. These incompatibilities with lactose are often produced by catalytic decomposition, known as the

Maillard reaction [13, 14]. This reaction may occur on compounds known as calistegines, as reported for the extract of

Physalis peruviana, which are tropane alkaloids and have secondary amines in the structure [29].

The results of similarity factor with corn starch, microcrystalline cellulose and polyvinylpyrrolidone (PVP), showed that

all the profiles observed in the ranges of wavelength evaluated are similar to the extract. This behavior shows the

compatibility between the extract and these excipients.

3.3. Development of a dry powder formulation

Due to the technological problems associated with the dry extract, caused by inappropriate pharmaceutical (physical-

mechanical) characteristics related to the nature of the extract as a multicomponent system, the fluid extract was absorbed
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in a substrate. This operation involves physical state changes that could help improve the different pharmaceutical

properties. According to the compatibility analysis performed (see table 3), some of the excipients that showed

compatibility with the extract were selected for this study. The assays were performed according to the SED described in

table 1, and the results of the response variables are shown below.

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Physical-mechanic properties

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Size and particle size distribution

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When performing a dry powder formulation of an extract, which involves absorption onto a substrate, it is necessary to

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obtain particle sizes that are fine to moderately thick, with less than 10% of fine particles. These aspects are important to

facilitate the subsequent processes, such as the mixing operation involved in the formulation and manufacture of the solid
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dosage form [30].
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Volume-surface diameter (Dvs) is the most important measure of the central tendency of the particle size, and other

parameters such as specific surface area and specific particle volume weight mean can be obtained from this measurement
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[20]. According to the particle size classification given by the USP 38 [19] , table 4 shows the particle sizes obtained by
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each formulation. The absorbates prepared with corn starch (A), microcrystalline cellulose PH 101 (B) microcrystalline
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cellulose PH 102 (C) and the mixture of microcrystalline cellulose PH 102, corn starch and PVP (F) have a particle size

from coarse to very thick, while those made from microcrystalline cellulose PH 200 (D) and the mixture of

microcrystalline cellulose and corn starch (E) have a particle size between coarse and moderately coarse. According to
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these results, the formulations D and E showed suitable behavior for the purposes of the study [30, 31].

In general, the particle sizes resulting from the formulations were larger than those of carriers, probably due to the extract,

which acts by binding the particles in each material; this effect is greater in materials that have a smaller particle size,

because they have a larger surface area (see table 4) [18, 32].

**** Table 4 ****

According to the particle size distribution, materials A, B and F have an average particle size between 850 and 2000

microns, and can be classified a thick to very thick. On the other hand, materials C and E have an average size of between
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425 and 850 microns, and can therefore be classified as moderately thick to thick, finally for material D, the average size

is between 250 and 425 microns and it is classified as between moderately fine and coarse.

The content of fine particles is greater for solid materials dried in the fluid bed (A-2, B-2, C-2, D-2, E-2 and F -2) than for

those procesed in the circulating air oven or static bed (A-1, B-1, C-1, D-1, E-1 and F-1). These results are a consequence

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of the method used, since the turbulence in the fluidized bed could cause excessive wear, breaking some large particles

and resulting in fine particles during the process [32, 33]; more detail information can be found in supplementary content

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(Fig. S1).

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The main characteristic desired in the formulations is that the percentage of fine particles does not exceed 10%. This

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feature is found in almost all the dry powders, except those with microcrystalline cellulose PH 200 (assays D-1 and D-2).

According to the previous discussion, the content of fine particles remains within the acceptable range, independent of the
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method used to produce the formulations, except the case of dry powders prepared with microcrystalline cellulose PH

200.
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Bulk and tap densities

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The bulk and tap densities of materials are directly related to their particle size and particle packing. These properties are
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crucial because they give an idea of the compactability of the materials and their cohesion characteristics [20, 32].
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According to the results in Fig. 2, the materials recommended as excipients are microcrystalline cellulose PH 200 (D) and

corn starch (A). Similarly, the formulations containing mixtures of these two materials would be suitable because of their

high density and therefore, low bulk. This behavior can be explained by the fact that the apparent volume is the total

volume of the powder, where the volume of the mass and the extraparticular spaces are included [20, 30, 32]. A high bulk

density value indicates better accommodation of the particles according to their size, i.e. the particles can fill a volume, to

produce a powder which is in static equilibrium due to the interaction of gravitational and adhesion/cohesion forces [20,

30, 32].

The results of this test demonstrate an increase in tap density in all cases, taking bulk density as reference. The change in

the volume, which affects the density, is caused by a rearrangement of the geometry of compaction in the bed of the
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particles, generated by transitions from loosely packed particles, in a more compact form, i.e. the cohesive forces between

particles increases [20, 32].

**** Figure 2 ****

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Based on the results obtained for the Carr index (Supplementary content Fig. S2), the formulations made from the

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cellulose (tests B, C and D) have a flow of excellent to good [20, 32], independent of the drying equipment used. The dry

powders made from starch (test A) have a flow of good to acceptable. The combinations between cellulose and starch

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(tests E and F) have an intermediate flow [20, 32].

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Angle of repose
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The flow properties of dried products are directly related to their behavior during storage, handling, and processing.

According to USP 38-NF 33 [19] and the results shown in the Fig. 3, the angle of repose of each formulation is between
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30 - 50, i.e. they which can be classified as materials with poor flow, except for the absorbates from corn starch, which
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can be classified as having excellent flow [20, 32].

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****Figure 3****
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Starch generally has high cohesiveness, and its flow characteristics are poor, however the flow properties also depend on
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the moisture content and after thorough drying, a free flowing material can be obtained [18]. During the preparation of

this formulation, it was possible to obtain a powder with better flow properties, partly due to the agglutination of the

particles in the presence of the extract, and partly due to the drying methods used, which allowed moisture content below

5% to be obtained.

Microcrystalline cellulose fluidity will depend not only on the moisture content, but also on the size and shape of the

particles and the content of fine particles. Microcrystalline cellulose PH 200 provides better flow than PH 101 and PH

102, since the former has a more spherical shape and a larger particle size [18]. Experimentally, it was found that the

formulations with microcrystalline cellulose PH 102 and PH 200 had a lower flow compared to the absorbate PH 101.

This is possibly due to the greater absorbtion capacity of microcrystalline celluloses PH 102 and PH 200, which causes

the cohesiveness between the particles to increase, and therefore, the fluidity of these absorbates to decrease.
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Hygroscopicity

According to the results shown in Fig.4 and the classification given by Callahan et.al [23], all the formulations were

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slightly hygroscopic, and increases in the moisture content above 10% were not produced when they were exposed to

environments with humidities below 80%. When the RH of the chamber was greater than 80%, the increase in moisture

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content of the dry powders after storage for one week was less than 40%, therefore these formulations could be classified

as slightly hygroscopic (class II). Most solid materials used in the pharmaceutical field are class I or class II, which favors

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their physical stability under different environmental conditions, requiring only standard containers [23].

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****Figure 4****
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The dry extract showed class III behavior because the moisture content does not increase above 5% after storage at

relative humidities below 60%. The increase in moisture content after storage for one week above 80% RH is less than
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50% [23].
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Detailing each formulation, it can be inferred that those made from starch have better performance against moisture,
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compared with those prepared with the different celluloses, as these only gained between 22% and 27% of moisture. For
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this reason, optimum performance of the excipients of the absorbent is required, to improve the behavior of the

formulation against moisture when a significant reduction of the hygroscopicity of the extract of fruits of Physalis
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peruviana is needed; this could be done by preparing mixtures of microcrystalline cellulose and small proportions of

starch, which does not affect the absorbent ability of the microcrystalline cellulose. As the results for the different

evaluated properties show, formulations made with mixtures exhibit an intermediate response with respect to the

individual materials used (Supplementary content Fig. S3).

Comparing the results of the behavior against moisture in the dry extract and the formulations (Fig. 4), there is evidence

that the transformation in the solid state of the extract of Physalis peruviana enables a better performance of this against

moisture, moving from class III to class II, even avoiding deliquescence of the extract, due to the low proportion of extract

in the formulation and the high absorbent capacity of the materials used for this purpose.

According to the ANOVA analysis of each variable evaluated, a statistically significant difference between the materials

and the equipment used to manufacture the absorbates was found. In order to show which factors may account for this
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difference, a statistical comparison of mean variables response measurements was performed, by means of the comparison

test. It was found that there are only statistically significant differences between the formulations made from

microcrystalline cellulose PH 102 and PH 200 in the oven and in the fluid bed. This is because there is usually better

fluidity of absorbates dried in the fluid bed than in the circulating air oven, a fact that is directly attributed to the drying

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method used; the fluidizing material enables particles with a more spherical shape to be obtained.

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The results assessed in this study are summarized in Table 5. According to the statistical analysis, it can be seen that

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almost all formulations are similar and therefore it is possible to select one of them based on the expected results. Besides,

the statistical difference found between the formulations with microcrystalline cellulose PH 102 and PH 200 is not

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relevant to selecting the final formulation because their properties did not show the expected behavior. According to

expectations for a future phytotherapeutic solid dosage form, the aim was to look for a formulation with a particle size of

between 425 and 850 um, a percentage of fine particles lower than 10%, and bulk and tap densities as high as possible,
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with a change of density greater than 20%. Regarding flowability and the Carr’s Index, a value between 12 and 21 is

expected, and a repose angle between 30 and 60 degrees. With regard to hygroscopicity, a classification class I or class II
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is expected. It was found that the material that best meets the expected parameters is E-2, i.e. the combination of
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microcrystalline cellulose PH 102 and corn starch. This combination is therefore suggested, as an option for the

development a dry powder formulation from ethanol extract of fruits of Physalis peruviana L.
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****Table 5****
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3.4. Hypoglycemic assay

The results in Fig. 8 show the hypoglycemic assay, where at time zero of the experiment, all the animals had low glucose

levels and were not statistically different, indicating that each population of animals has an homogeneous BGL. Between

one hour and two hours of the assay, maximum BGL were reached in the control group. All the mice recovered normal

BGL, thanks to their own homeostasis. Based on these results it appears that the excipients of the absorbate do not

interfere with the assay, and also the dry powder formulation of the extract retains its hypoglycemic activity, suggesting

that it is possible to continue with the subsequent development processes of a phytotherapeutic product based on ethanol

extract of fruits from Physalis peruviana L.

****Figure 5****
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3. Conclusions

Microcrystalline cellulose, corn starch and PVP were selected from the compatibility studies to develop a dry powder

formulation from Physalis peruviana L. This formulation presents better characteristics in terms of particle size, density,

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voluminosity, flowability and hygroscopicity, compared with the extract. The hypoglycemic assay allowed establishing

the transformation to an oral solid dosage form of the extract did not have a significant effect against its pharmacological

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activity, indicating that this formulation is a good option for obtaining a future phytotherapeutic product or an

intermediate herbal product from ethanol extract of fruits of Physalis peruviana, for the adjuvant treatment of diabetes.

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Acknowledgements: The authors acknowledge the Research Division of Universidad Nacional de Colombia (Project

Code: 15192), Ministerio de Agricultura y Desarrollo Rural, ASOHOFRUCOL and Laboratorios Bussié S.A. for the
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financial support of this work.
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4. References

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[2] CBI Market Survey, The market for natural ingredients for pharmaceuticals in the EU, (2008).
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[4] N. Sharapin, L. Machado Rocha, E.S. Carvalho, Fundamentos de tecnología de productos fitoterapéuticos, Convenio Andrés Bello :

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CYTED, Santafé de Bogotá, 2000.
[5] J. Correa, H. Bernal, Especies vegetales promisorias de los países del convenio Andrés Bello, Santafe de Bogotá, Colombia, 1992.
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fruit: A review, Food Research International, 44 (2011) 1733–1740.

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[7] H. García Barriga, Flora medicinal de Colombia botánica médica, Instituto de Ciencias Naturales, Universidad Nacional de Colombia,
Bogotá, Colombia, 1974.
[8] D.P. Rey, L.F. Ospina, A.D. Marcela, Inhibitory effects of an extract of fruits of Physalis peruviana on some intestinal

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carbohydrases., Revista Colombiana de Ciencias Químico Farmacéuticas, 44 (2015) 72-89.
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[10] R.O.B. Wijesekera, The medicinal plant industry, CRC Press, Boca Raton, 1991.
[11] P.H. List, P.C. Schmidt, Phytopharmaceutical technology, CRC Press, Boca Ratón, Florida, 1989.
[12] D. Rodriguez, M. Espitia, Y.E. Caicedo, Y.E. Cordoba, Y. Baena, C.E. Mora, Caracterización de algunas propiedades fisicoquímicas

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y farmacotécnicas del almidón de arracacha (Arracacia xanthorriza), Revista Colombiana de Ciencias Químico Farmacéuticas, 34 (2005)
140-146.
[13] S.H. Kopelman, L.L. Augsburger, Excipient compatibility study of Hypericum perforatum extract (St. John's Wort) using similarity
metrics to track phytochemical profile changes, International Journal of Pharmaceutics, 237 (2002) 35-46.
[14] N. Wyttenbach, C. Birringer, J. Alsenz, M. Kuentz, Drug-excipient compatibility testing using a high-throughput approach and
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statistical design, Pharmaceutical Development And Technology, 10 (2005) 499-505.
[15] A.T. Serajuddin, A.B. Thakur, R.N. Ghoshal, M.G. Fakes, S.A. Ranadive, K.R. Morris, S.A. Varia, Selection of solid dosage form
composition through drug-excipient compatibility testing, Journal of Pharmaceutical Sciences, 88 (1999) 696-704.
[16] Y. Wu, M. Dali, A. Gupta, K. Raghavan, Understanding drug-excipient compatibility: oxidation of compound A in a solid dosage
form, Pharmaceutical Development And Technology, 14 (2009) 556-564.
[17] D.P. Medina, Implementación de una metodología para la obtención de marcadores de frutos de Physalis peruviana L.
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y evaluación de la actividad hipoglicemiante, Facultad de Ciencias, Departamento de Farmacia, Universidad Nacional de Colombia,
Bogotá, Colombia, 2012.
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[18] R.C. Rowe, P.J. Sheskey, S.C. Owen, A.P. Association., Handbook of pharmaceutical excipients, 6th ed., Pharmaceutical Press;
American Pharmacists Association, London; Chicago, 2009.
[19] United States Pharmacopeia and National Formulary, USP 38–NF 33, The United States Pharmacopeial Convention, Rockville, MD,
2015.
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[20] A.N. Martin, P.J. Sinko, Y. Singh, Martin's physical pharmacy and pharmaceutical sciences physical chemical and
biopharmaceutical principles in the pharmaceutical sciences, 6th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2011.
[21] S.D. Palma, R.H. Manzo, D.A. Allemandi, Dry plant extracts loaded on fumed silica for direct compression: preparation and, Pharm
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Dev Technol, 4 (1999) 523-530.

[22] S. Palma, C. Luján, J.M. Llabot, G. Barboza, R.H. Manzo, D.A. Allemandi, Design of Peumus boldus tablets by direct compression
using a novel dry plant extract, International Journal of Pharmaceutics, 233 (2002) 191-198.
[23] J.C. Callahan, G.W. Cleary, M. Elefant, G. Kaplan, T. Kensler, R.A. Nash, Equilibrium Moisture Content of Pharmaceutical
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Excipients, Drug Development and Industrial Pharmacy, 8 (1982) 355-369.

[24] L. Gallo, M.V. Ramirez-Rigo, J. Piña, S. Palma, D. Allemandi, V. Bucalá, Valeriana officinalis Dry plant extract for direct
compression: Preparation and characterization, Scientia Pharmaceutica, 80 (2012) 1013-1026.
[25] L.F. Ospina, R. Pinzón, Plantas Usadas Como Antidiabéticas en la Medicina Popular Colombiana, Revista Colombiana de Ciencias
Químico Farmacéuticas, 23 (1995) 81-94.
[26] A.C. Mora, D.M. Aragón, L.F. Ospina, Effects of Physalis peruviana Fruit Extract on Stress Oxidative Parameters In Streptozotocin-
Diabetic Rats, Latin Americam Journal of Phramacy, 29 (2010) 1132-1136.
[27] K.A. Connors, G.L. Amidon, Chemical stability of pharmaceuticals a handbook for pharmacists, John Wiley, New York, 1986.
[28] J.L. Sims, J.A. Carreira, D.J. Carrier, S.R. Crabtree, L. Easton, S.A. Hancock, C.E. Simcox, A new approach to accelerated drug-
excipient compatibility testing, Pharmaceutical Development And Technology, 8 (2003) 119-126.
[29] N. Asano, A. Kato, K. Matsui, A.A. Watson, R.J. Nash, R.J. Molyneux, L. Hackett, J. Topping, B. Winchester, The effects of
calystegines isolated from edible fruits and vegetables on mammalian liver glycosidases, Glycobiology, 7 (1997) 1085-1088.
[30] R. Voigt, M. Bornschein, Tratado de tecnología farmacéutica, 3 ed., Acribia, Zaragoza, 1982.
[31] J.P. Remington, D.B. Troy, Remington the science and practice of pharmacy, 21th. ed., Lippincott Williams & Wilkins,
Philadelphia, 2006.
[32] M.E. Aulton, Aulton's pharmaceutics the design and manufacture of medicines, 3rd ed., Churchill Livingstone, Edinburgh New
York, United States, 2007.
[33] K.L.E. Kadam, Granulation technology for bioproducts, CRC Press, Boca Ratón, Florida, 1991.
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2

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Fig. 1 Chromatographic profiles of the Physalis peruviana extract at 280 nm mixed with
different excipients
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Conventions: 1 = Negative control (Physalis peruviana extract); 2 = Dicalcium phosphate;

3 = PVP K30; 4 = Microcrystalline cellulose; 5 = Corn starch; 6 = Lactose.
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0.70

0.60

0.50

Density g/ml
0.40

0.30

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0.20

0.10

0.00
A-1 A-2 B-1 B-2 C-1 C-2 D-1 D-2 E-1 E-2 F-1 F-2

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Formulation
Bulk density Tap density

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Fig. 2 Bulk an tap densities of the formulations.
A: Corn starch, B: microcrystalline cellulose PH 101, C: microcrystalline cellulose PH 102,
D: microcrystalline cellulose PH 200, E: microcrystalline cellulose PH 102 + corn starch,

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F: microcrystalline cellulose PH 102 + corn starch agglutinated with PVP K 30; 1: Air oven
2: Fluidized bed. MA
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50
45
40
35

Angle of repose

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30
25
20
15

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10
5
0

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A-1 A-2 B-1 B-2 C-1 C-2 D-1 D-2 E-1 E-2 F-1 F-2
Formulation

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Fig. 3 Angle of repose of the formulations..
A: Corn starch, B: microcrystalline cellulose PH 101, C: microcrystalline cellulose PH 102,
D: microcrystalline cellulose PH 200, E: microcrystalline cellulose PH 102 + corn starch,
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F: microcrystalline cellulose PH 102 + corn starch agglutinated with PVP K 30; 1: Air oven
2: Fluidized bed.
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45%

40%

35%

30%

Weight change (%)

25%

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20%

15%

10%

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5%

0%
0 50 100 150 200 250 300

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-5%
Time (h)
38% EX 52% EX 72% EX 97% EX
38% F 52% F 72% F 97% F d

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Fig. 4 Behavior of the extract (EX) and the formulation selected E-2 (F) against moisture
(38%, 52·%, 72% and 97% RH) MA
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Fig. 5 Hypoglycemic activity.

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Average  S.D.; n= 10 animals per group; Mice ICR. ** p0.01, ***p0.001 Bonferroni
test.
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Table 1
Statistical Experimental Design for the dry powder formulation of the extract from fruits of Physalis peruviana L.

Experiments Equipment 1 (air oven) Equipment 2 (fluidized bed)

1 A C
2 B D
3 C E
4 D F

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5 E A
6 F B

A: Corn starch, B: microcrystalline cellulose PH 101, C: microcrystalline cellulose PH 102, D: microcrystalline cellulose PH 200, E: microcrystalline

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cellulose PH 102 + corn starch, F: microcrystalline cellulose PH 102 + corn starch agglutinated with PVP K 30.

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Table 2
Treatment groups in testing oral hypoglycemic activity in starch overload assay.

Excipients
Positive
Control group (Corn starch and microcrystalline Formulation
control group
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cellulose)

Oral overload Starch USP 2000 mg / Kg

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Treatment Glycerin: Propylene glycol: Equivalent extract

Acarbose (20mg / Kg) (500 mg of excipients mix/ kg)
water (20:20:80) (500mg / kg)
(n= 10 animals per group)
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Table 3
Similarity test results to compare the chromatographic profiles.

Excipient wavelength (nm) f2 Excipient Wavelength (nm) f2

210 52,97 210 56,15
254 76,42 Microcrystalline 254 51,94
Dibasic calcium phosphate
280 42,76 cellulose 280 95,46

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350 95,68 350 91,09
210 42,44 210 56,62
254 51,15 254 57,46
Lactose Starch
280 69,24 280 72,26

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350 89,51 350 86,67
210 57,97
254 90,42
PVP
280 98,32

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350 79,49

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Table 4
Average particle size and particle size distribution of each one of the formulations.

Particle size distribution (µm)*

Material DVS (µm)
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1425 637.5 337.5 215

A-1 1333,03 55.42% 32.48% 9.49% 1.79%
A-2 1357,84 64.31% 27.35% 5.60% 2.11%
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B-1 1363,02 65.61% 24.78% 7.57% 1.28%

B-2 1282,71 43.38% 42.92% 10.96% 2.03%
C-1 1190,41 23.18% 30.80% 40.96% 3.12%
C-2 1076,23 16.08% 49.13% 28.89% 4.97%
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D-1 669,83 1.61% 22.47% 44.77% 22.88%

D-2 474,54 0.23% 8.58% 48.01% 22.57%
E-1 871,31 5.37% 42.82% 45.57% 4.34%
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E-2 843,58 5.99% 41.41% 42.57% 6.85%

F- 1 1352,12 43.41% 43.05% 9.38% 1.17%
F-2 1350,46 57.27% 24.51% 10.72% 2.23%

* These values represent the percentage of total weight of material retained in each sieve.
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A: Corn starch, B: microcrystalline cellulose PH 101, C: microcrystalline cellulose PH 102, D: microcrystalline cellulose PH 200, E: microcrystalline
cellulose PH 102 + corn starch, F: microcrystalline cellulose PH 102 + corn starch agglutinated with PVP K 30; 1: Air oven 2: Fluidized bed.
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Table 5
Summarized results of all measured variables.

voluminos
Change in

Change in
size (µm)

Fines (%)

Hygrosc
Particle

Angle of

opicity
density

density

density

Carr’s
ity (%)
(g/mL)

(g/mL)

repose
index
Bulk

Tap

(%)
the

the
Variable

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Class I or
Reference value 425 - 850 < 10 > > >20% >20% 12 - 21 30 -60
class II
Average 1357,84 1,95% 0,48 0,59 22,90 -18,18 17,95 28,48 Class II
A1
Deviation - 0,01 0,025 0,026 - - 6,549 1,590 -
Average

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1333,03 2,41% 0,47 0,59 25,53 -20,28 20,38 24,37 Class II
A2
Deviation - 0,01 0,015 0,019 - - 3,954 5,041 -
Average 1282,71 1,60% 0,32 0,38 18,75 -13,60 13,73 36,13 Class II
B1
Deviation - 0,01 0,014 0,021 - - 4,302 3,141 -

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Average 1363,02 2,35% 0,29 0,34 17,24 -15,42 15,42 35,17 Class II
B2
Deviation - 0,02 0,011 0,012 - - 3,858 2,722 -
Average 1076,23 4,34% 0,37 0,41 10,81 -9,66 9,87 47,09 Class II
C1
Deviation - 0,01 0,018 0,009 - - 3,422 2,534 -

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Average 1190,41 5,44% 0,27 0,33 22,22 -10,32 17,73 41,47 Class II
Formulation

C2
Deviation - 0,03 0,009 0,016 - - 2,788 2,270 -
Average 474,54 30,71% 0,44 0,49 11,36 -10,53 10,65 42,24 Class II
D1
Deviation - 0,08 0,011
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Average 669,83 42,78% 0,46 0,51 10,85 -10,50 10,29 41,46 Class II
D2
Deviation - 0,10 0,018 0,028 - - 2,291 3,132 -
Average 871,31 5,14% 0,36 0,43 19,44 -16,66 16,48 36,08 Class II
E1
Deviation - 0,02 0,027 0,014 - - 6,000 4,333 -
Average 843,58 9,71% 0,35 0,44 24,71 -20,68 20,64 35,51 Class II
E2
Deviation - 0,01 0,015 0,015 - - 1,893 4,374 -
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Average 1352,12 1,36% 0,34 0,43 26,47 -20,87 20,43 42,68 Class II
F1
Deviation - 0,01 0,04 0,020 - - 7,615 1,556 -
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Average 1284,46 2,54% 0,34 0,43 26,47 -22,14 21,57 38,36 Class II
F2
Deviation - 0,02 0,04 0,019 - - 6,955 1,029 -
A: Corn starch, B: microcrystalline cellulose PH 101, C: microcrystalline cellulose PH 102, D: microcrystalline cellulose PH 200, E: microcrystalline
cellulose PH 102 + corn starch, F: microcrystalline cellulose PH 102 + corn starch agglutinated with PVP K 30; 1: Air oven 2: Fluidized bed.
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Graphical abstract

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Highlights

 A study of the compatibility between the excipients and the

extract was conducted.
 The extract was formulated in oral solid dosage form.

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 Some powder characteristics of the formulation were
evaluated.

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 The hypoglycemic activity remained after transformation to

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solid state.

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