Phytomedicine Plus 2 (2022) 100337

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Phytomedicine Plus
journal homepage: www.sciencedirect.com/journal/phytomedicine-plus

In-vivo evaluation and formulation development of polyherbal extract in

streptozotocin-induced diabetic rat
Sagarika Majhi a, *, Lubhan Singh b, Madhu Verma a, Iti Chauhan a, Raj kumari a,
Meenakshi Sharma a
a
I. T. S College of Pharmacy, Muradnagar, Ghaziabad, India
b
Kharvel Subharti College of Pharmacy, Swami Vivekanand Subharti University, Meerut, India

A R T I C L E I N F O A B S T R A C T

Keywords: Background: The research focuses on polyherbal antidiabetic extracts from various plants that are utilized in the
Polyherbal (PH) formulation management of type 2 diabetes mellitus at various doses. Ayurvedic treatment is widely accepted throughout due
Streptozotocin to their efficacy, safety, less price, ubiquity, and acceptance. Polyherbal medicines, contain glycosides, alkaloids,
Diabetes mellitus
flavonoids, and other compounds that have unique mode of action to treat diabetes, have long been used across
Antihyperlipidemic parameters
the world. This study was carried out to investigate the hypoglycemic effects and antihyperglycemic activity of
Antioxidant activity
Tablet polyherbal (PH) extract in streptozotocin (STZ)-induced diabetic rats. Along with this, the antioxidant and
Blood glucose level (BGL) antihyperlipidemic activity of Polyherbal extract (PH) were also studied. Further, polyherbal tablets were
formulated and evaluated.
Method: Polyherbal extract (hydroalcoholic) was tested for the hypoglycemic activity for 4 h in normoglycemic
rats, oral glucose tolerance test (OGTT) (2 h), and antihyperglycemic activity (28 days) in non-diabetic and STZ-
induced diabetic rats. Further, antihyperlipidemic and antioxidant activity were also studied along with histo­
pathology and pre & post-compression tablet data. The effective antidiabetic activity was observed for PH I (400
mg/kg followed 200 mg/kg). OGTT has shown that PH I (200 mg/kg) extract is having a better glucose utili­
zation capacity. The data also propose that PH I (400 mg/kg followed 200 mg/kg) exhibit significant and
consistent hypoglycemic activity.
Results: Our findings explain that polyherbal extract in variant doses has antihyperlipidemic and antioxidant
activity, which is manifested by decreasing total cholesterol, triglyceride, LDL, VLDL and MDA level. The extract
also increased the HDL, SOD and reduced glutathione level in various treatment groups. The data propose that
the combined extract showed a consistent and ameliorated hepatic and renal function, in the treatment groups.
Polyherbal extract PH I (400 mg/kg) have manifested the most effective improvements in diabetic complications.
All the pre & post-compression tablet data were within limits. The above results propose that this Polyherbal
extract may be a potential source for the development of the new antidiabetic drug.
Conclusions: The proposed activity of the polyherbal extract is supported by various biochemical and histo­
pathologic analyses. By examining its safety and in vivo efficacy, the combination of ayurvedic components will
give us a novel glimmer of hope for the lifestyle illness.

1. Introduction
Diabetes mellitus (DM) is an earnest, persistent ailment that occurs
when either one’s body cannot use the produced insulin efficiently
(hormone regulating blood glucose level), or if the pancreas does not
process adequate insulin. It is a salient public health issue, earmarked as
a target by health sector experts. Early diagnosis is the beginning point
for living well with the disease, as the longer, a person is untreated and
undiagnosed, the more the consequences are likely to be. In India, the
etiology behind diabetes is multifactorial as it incorporates genetic
factors along with obesity, lifestyle changes, and rising urban migration
(Wild et al., 2004; Anjana et al., 2011). Easy ingress to basic facilities,
like blood glucose or HbA1c testing (glycated hemoglobin test), should
therefore be readily available in healthcare organizations. As

* Corresponding author.
E-mail address: sagarika.majhi@gmail.com (S. Majhi).

https://doi.org/10.1016/j.phyplu.2022.100337

Available online 5 September 2022

2667-0313/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
S. Majhi et al. Phytomedicine Plus 2 (2022) 100337

Table 1 activity of the chemical constituents such as alkaloids, flavonoids,

Constituents of polyherbal formulation (pH I). phenolics, triterpenoids, xanthones etc. has been reported. All the herbs
Plant Name & Extract Extract Extract Yield Dose of used in the study individually possess potent hypoglycemic or anti­
Authentication Voucher Yield (%) Dose (mg/ (mg)/ 100 g of Powder hyperglycemic or antioxidant activity. The medicinal plants used in
No. kg) Powder (g) (g)/ study was based on their previous established pharmacological actions,
Kg Body
summarized as follows:
wt.

Aloe vera 35 130 35,000 0.37 a) Aloe vera (L.) Burm.f. (Leaf pulp) (Hypoglycemic activity - Yagi
(MCM/Bot-2/2012)
Camellia sinensis 5.5 100 5500 1.82
et al., 2009)
(MCM/Bot-3/2012) b) Camellia sinensis (L.) Kuntze (Leaves) (Antihyperglycemic activity
Capparis decidua 15 500 15,000 3.33 - Gomes et al., 1995)
(NHCP/NBPGR/ c) Musa sapientum (L.) (Flower) (Hypoglycemic activity - Pari and
2012–15/5317)
Maheswari, 1999).
Musa sapientum 2.61 10 2610 0.38
(MCM/Bot-5/2012) d) Tinospora cordifolia (Willd.) Miers (Root) (Hypoglycemic activity -
Phyllanthus amarus 21.5 600 21,500 2.79 Stanely et al., 2000)
(MCM/Bot-7/2012)
Punica granatum Flower 40.1 400 40,100 1.00
(NHCP/NBPGR/
2012–16/5320) Table 3
Punica granatum Seed 21.5 200 21,500 0.93 Experimental design.
(NHCP/NBPGR/
2012–17/5321) Groups Route & Dose of Drug BGL at Day 0
Tinospora cordifolia 2.3 5000 2300 217.39 Group I Normal Orally with vehicle (1 mL/kg BW) 90.45 ± 1.50
(NHCP/NBPGR/ Control
2012–18/5322) Group II Diabetic Orally with vehicle (1 mL/kg BW) 256.95 ±
Control 2.12#
Group Standard Orally with Glibenclamide (600 μg/ 280.94 ±
periodically patients need assessment or treatment for complications by III kg BW) 2.25
the specialists, entrenched systems for referral and back-referral are Group Test drug Orally with PH I (200 mg/kg BW) 282.70 ±
IV 3.44
needed. To yield better outcomes, emphasis is given to improve the
Group V Test drug Orally with PH I (400 mg/kg BW) 295.91 ±
diagnosis and treatment of diabetes (WHO, 1999). A variety of antidi­ 3.08
abetic drugs are available in the market, but they often produce unde­
sirable side effects like abnormally low sugar, lactic acidosis, abdominal
malaise etc. after prolonged consumption. The current exploration was Table 4
designed to assess the probable beneficial effects of these natural herbs Standardization parameters of polyherbal tablet.
in combination on blood glucose level of streptozotocin (STZ)-induced
Organoleptic Characters
diabetic rats, which are commonly consumed along with the diet. In
1. color Brown
Ayurveda, the drug formulation is based on two principles:
2. odor Characteristic
1. Using more than one herb for the formulation is known as Poly­ 3. Taste Bitter
herbal (PH) formulation. It utilizes a combination of herbs to achieve Physicochemical Parameters
therapeutic effectiveness (Little, 2009). 1. PH 7.8
2. PH has a wide therapeutic index i.e., effective at a low dose and 2. Moisture content 1.02%
3. Loss on drying 2.14%
safe at high dose, (better risk: benefit ratio) (Joshi et al., 2007) as
4. Total ash 5.43%
compared to allopathic hypoglycaemic agents having narrow thera­ 5. Acid-insoluble ash 1.44%
peutic range. pH is ideal for treatment purposes due to their efficacy, 6. Water-soluble ash 3.23%
safety, less price, ubiquity, and acceptance. PH can exert the best ther­ 7. Water-soluble extractive value 14.98%
8. Ethanol-soluble extractive value 12.34%
apeutic effect on human health by correct and rational use (Jawla et al.,
9. Arsenic not more than 5 ppm Complies
2009; Chatterjee et al., 2012). The occurrence of DM is surging in the 10. Lead not more than 10 ppm Complies
population and forcing a financial burden on the healthcare system as Post Compression Parameter
well as individuals suffering from the disease. It imposes a threat on 1. Hardness (kg/cm2) 5.86 ± 0.29
quality of life as remains undiagnosed in many patients and the overall 2. Thickness (mm) 4.11 ± 0.12
3. Friability (%) 0.53%
cost of healthcare to treat diabetes is also important. Thus, some
4. Average weight (mg) 351.67
definitive solutions are required which depend on the challenges for 5. Disintegration time 15.57 ± 0.91
point prevention and treatment in new directions. The antidiabetic

Table 2
Composition of polyherbal tablet.
Batch Weight of granule Na CMC (mg) MCC SSG Magnesium stearate Starch Lactose Anhydrous Total weight of Tablet
(mg) (mg) (mg) (mg) (mg) (mg) (mg)

B1 228 20 – – 15 50 q. s 350
B2 228 30 – – 15 50 q. s 350
B3 228 40 – – 15 50 q. s 350
B4 228 – 20 – 15 50 q. s 350
B5 228 – 30 – 15 50 q. s 350
B6 228 – 40 – 15 50 q. s 350
B7 228 – – 20 15 50 q. s 350
B8 228 – – 30 15 50 q. s 350
B9 228 – – 40 15 50 q. s 350

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S. Majhi et al. Phytomedicine Plus 2 (2022) 100337

Table 5
Pre-compression study data.
Batch Bulk density (g/cm3) Tapped density (g/cm3) Angle of repose (θ) Carr’s index Hausner’s ratio

B1 0.411 ± 0.004 0.468 ± 0.006 29 ± 0.88 12.17 ± 0.90 1.14 ± 0.02

B2 0.511 ± 0.004 0.577 ± 0.003 32 ± 0.88 11.49 ± 0.83 1.13 ± 0.02
B3 0.571 ± 0.005 0.641 ± 0.004 34 ± 1.16 10.90 ± 0.86 1.12 ± 0.02
B4 0.537 ± 0.004 0.610 ± 0.003 32 ± 1.16 12.00 ± 1.04 1.14 ± 0.02
B5 0.556 ± 0.006 0.652 ± 0.005 34 ± 1.16 14.80 ± 0.97 1.17 ± 0.02
B6 0.538 ± 0.005 0.634 ± 0.007 30 ± 0.88 15.12 ± 0.66 1.18 ± 0.02
B7 0.530 ± 0.010 0.630 ± 0.010 33 ± 0.88 15.88 ± 0.25 1.19 ± 0.01
B8 0.539 ± 0.006 0.616 ± 0.004 34 ± 1.20 12.45 ± 0.66 1.14 ± 0.01
B9 0.537 ± 0.011 0.627 ± 0.004 34 ± 1.16 14.34 ± 1.60 1.17 ± 0.04

Fig. 1. Effect of PH I on the Bodyweight

Results are displayed as mean ± SEM
(n = 6 rats/group);
*P < 0.01; when Diabetic control, PH I
(200 mg/kg) compared with their
respective initial body weight (Paired t-
test) (NC–Normal Control; DC-Diabetic
Control; STD-Standard Glibenclamide;
PH I Polyherbal formulation I).

As per the literature survey and documented research evidences, the

Table 6
current polyherbal combination was prepared so as to achieve a better
Effect of PH I on Food, and Water Intake of STZ induced rats. therapeutic alternative for diabetic patients. The medical professionals
are grappling alternative medications along with routine therapeutic
Groups Food Intake (g/Day) Water Intake (ml/Day)
management to manage diabetes without causing any negative effects.
Control 49.61 ± 2.67 210.86 ± 7.91 Polyherbal remedies, have long been utilised to cure ailments in Asian
Diabetic Control 68.07 ± 2.36# 340.96 10.02#
±
cultures and across the world as they contain substances such as gly­
Glibenclamide (STD) 53.82 ± 1.69b 282.93 ± 9.06b
pH I (200 mg/kg) 58.14 ± 1.80a 300.93 ± 10.25a cosides, alkaloids, flavonoids, and other compounds that possess unique
pH I (400 mg/kg) 55.39 ± 2.17b 285.14 ± 8.94b mode of action to treat diabetes with low cost, safety and therapeutic
effectiveness. In the present study, various plant parts were used in
Results are displayed as mean ± SEM (n = 6 rats/group).
# P < 0.001; when Diabetic control compared with Normal control (Unpaired t-
combination as a polyherbal formulation. The formulation was used to
test). investigate their effect on blood glucose, lipid profile, serum creatinine,
a
P < 0.01; when PH I (200 mg/kg) compared with Diabetic control (1-Way SGOT, and SGPT in STZ induced model of diabetes mellitus in wistar
ANOVA, with Post Tukey’s test). bP < 0.001; when STD, PH I (400 mg/kg) albino rats.
compared with Diabetic control (1-Way ANOVA, with Post Tukey’s test).
2. Materials & methods
e) Capparis decidua (Forsk.) Edgew. (Fruits) (Antidiabetic activity -
Sharma et al., 2010) 2.1. Chemicals and reagents
f) Phyllanthus amarus Schumach. & Thonn. (Leaf) (Hypoglycemic and
hypocholesterolemic activities - Adeneye et al., 2006) Streptozotocin (STZ) was purchased from Hi-Media Laboratories,
g) Punica granatum Linn. (Flower) (Hypoglycemic activity - Jafri Mumbai. Glibenclamide was procured from Sigma-Aldrich. Various
et al., 2000) diagnostic kits were purchased from ERBA Diagnostic Mannheim
h) Punica granatum Linn. (Seeds) (Antidiabetic action - Das et al., (TRANSASIA Bio-medicals Ltd. Solan). All the chemicals and reagents
2001). employed for the study are of analytical grade.

3
S. Majhi et al.
2.2. Plant material collection & authentication
All the medicinal plants were recognized and collected from various
regional parts of India in August and September 2012. Further, the
plants were identified and authenticated at the National Bureau of Plant
Genetic Research (Dr. E Roshini Nayar, Principal Scientist, National),
New Delhi, and Chaudhary Charan Singh University (Dr. A K Gupta,
Reader, Department of Botany), Meerut. The plant voucher specimen
was deposited for reference in respective departments. The authentica­
tion voucher no. is provided in Table 1.
2.3. Preparation of herbal extract
The individual plant drugs were gathered and dried in shade at room
temperature at 37 ◦ C. Further, Camellia sinensis was coarsely powdered
by hand crushing and Aloe vera, Musa sapientum, Tinospora cordifolia,
Capparis decidua, Phyllanthus amarus, Punica granatum (Flower & seeds)
by grinding in the mixer grinder (SIKRI Herbs Grinder Machine HG001)
and then sieved manually. Each drug was weighed on a digital balance
and subjected to soxhlet extraction with 95% ethanol at 60–80 ◦ C
(before extraction of fruits, seeds, and stems, the process of defatting was
performed with petroleum ether at 60–80 ◦ C). The hydroalcoholic
extraction (ethanol:water 60:40) was carried out for 48 h (12 h/day), for
each batch of extract. The herb:solvent ratio was 1:5 and the yield were
depicted in Table 1. In the end, a rotary flash evaporator under vacuum
was used to concentrate the extract to a semi-solid mass with the re­
covery of solvent. The traces of the solvents were removed by using
Lyophilizer (S K Appliances PL-191). The resultant extract was stored at
4 ◦ C for future use.
2.4. Physicochemical properties of different plant extracts were performed
Various parameters viz. pH; Moisture content; Loss on drying; Ash
4
Phytomedicine Plus 2 (2022) 100337
Fig. 2. Effect of PH I in oral glucose
tolerance test on STZ-induced Dia­
betic rats
Results are displayed as mean ± SEM
(n = 6 rats/group).
# P < 0.01; when Diabetic control
compared with Normal control (Un­
paired t-test).
a
P < 0.001; when STD, PH I (200 &
400 mg/kg) compared with their
respective ‘0′ Minute (1-Way ANOVA,
followed by Post Tukey’s test
(NC–Normal Control; DC-Diabetic
Control; STD-Standard Glibencla­
mide; PH I Polyherbal formulation I).
values (Total ash; Acid-insoluble ash; Water-soluble ash); Water-soluble
and ethanol extractive values and trace materials (lead, arsenic) analysis
was used as a standardization tool of the plant material (The Ayurvedic
Pharmacopoeia of India, 2001; Ministry of AYUSH, 2021; Gupta, 2006).
2.5. Constituents of polyherbal (PH I) formulation
The Polyherbal (pH I) formulation was developed by mixing the
individual extracts (Table 1) i.e., Aloe vera (L.) Burm.f. (Leaf pulp) (Yagi
et al., 2009), Camellia sinensis (L.) Kuntze (Leaves) (Gomes et al., 1995),
Musa sapientum (L.) (Flower) (Pari and Maheswari, 1999), Tinospora
cordifolia (Willd.) Miers (Root) (Stanely et al., 2000), Capparis decidua
(Forsk.) Edgew. (Fruits) (Sharma et al., 2010), Phyllanthus amarus
Schumach. & Thonn. (Leaf) (Adeneye et al., 2006) and Punica granatum
Linn. (Flower) (Jafri et al., 2000) & Punica granatum Linn. (Seeds) (Das
et al., 2001) in concentration determined by below formula (Kumar
et al., 2015).
Dose of Powder per kg body weight (g) = [100 / Extract yield (mg) /
100 g of Powder] X Extract Dose (mg/kg)
2.6. Formulation of polyherbal tablets
The hydroalcoholic extract was adsorbed on lactose to get a free-
flowing powder. The granules were prepared by using isopropyl
alcohol. The granules were evaluated for various physical properties like
bulk density, tapped density, Hausner’s ratio, Carr’s index, and angle of
repose (Aulton, 2008; Ansel and Popovich, 2005. Tablets were prepared
using a single rotator tablet punching machine (10 stations), in which
the punch size was 11 mm × 8 mm. The composition of the tablets is
mentioned in Table 2, in which disintegrating agents (sodium carboxy
methyl cellulose, microcrystalline cellulose, and sodium starch glyco­
late) were used in different concentrations. Magnesium stearate was
used as a lubricant; starch was used as a binder and lactose anhydrous
S. Majhi et al. Phytomedicine Plus 2 (2022) 100337

Fig. 3. Effect of PH on Normoglycemic rats (Hypoglycemic effect)

Results are displayed as mean ± SEM (n = 6 rats/group).
#P>0.01 (not significant); when Normal control compared with its respective “0′′ min (1-way ANOVA, followed by Post Tukey’s test).
a
P<0.001; when STD, PH I (200 & 400 mg/kg) compared with their respective “0′′ min (1-way ANOVA, followed by Post Tukey’s test) (NC–Normal Control; DC-
Diabetic Control; STD-Standard Glibenclamide; PH I Polyherbal formulation I).

was used as a diluent for preparation of 350 mg tablets.
2.7. Evaluation of the formulated tablet
Tablets were evaluated for their general appearance like color, odor,
and taste along with thickness by using vernier calliper. Tablet’s hard­
ness and friability were measured using a dial-type hardness tester and
Roche friabilator respectively. The weight variation test was carried out
by weighing 20 tablets individually, calculating the average weight, and
comparing the individual tablet weight to the average. The disintegra­
tion time of the tablet was measured in water (37 ◦ C) using the USP
disintegration test apparatus (Indian Pharmacopoeia, 1996).
2.8. Experimental animal model selected for work
levels (5, 50, 300, and 2000 mg/kg body weight) dissolved in 1%
carboxymethylcellulose (CMC). One group was maintained as control
and administered the vehicle only. Then, the animals were observed
individually at least once during first 30 min, periodically during first
24 h (special attention for first 4 h), and daily thereafter for a total
period of 14 days. Furthermore, the behavior of animals and any other
toxic symptoms were observed and recorded systematically (individual
records being maintained for each animal).
2.8.3. Experimental design
Before the experiment, the selected animals were weighed and
grouped into five with six animals each as shown in Table 3. The com­
plete duration of the experiment was 28 days. Daily intake of food and
water was measured. Body weight was studied for all the groups in the 7-
day gap and on the last day of sacrifice.

2.8.1. Animal care and selection

2.8.3.1. Experimental induction of diabetes in rats. Adult albino rat
Albino rats, Wistar strain (150–200 g) and Swiss strain, Albino mice
(Wistar strain) weighing 150–200 g was selected for the study. Blood
(20–30 g) of either sex were obtained from the M.I.E.T animal house,
sample were collected for the determination of baseline glucose levels.
Meerut, Uttar Pradesh (India). Animals were housed under standard
DM was induced in overnight fasted animals. Rats were initially injected
conditions of temperature, humidity, and light: dark cycle of 25 ± 2 ◦ C,
with a single dose of nicotinamide (110 mg/kg body weight i.p) in
65 ± 10%, 12 h respectively. Standard food pellets and drinking water
physiological saline and 15 min later injected intraperitoneally with 65
was fed to animals ad libitum. The experimental protocol was approved
mg/kg of STZ freshly prepared in 0.01 M citrate buffer PH 4.5. Animals
by the IAEC of M.I.E.T, Meerut. All the animal activities were conducted
in control group received equivalent volume of the citrate buffer and are
according to the guidelines of “Guide for the care and use of laboratory
served as non-diabetic controls. After the elapse of 1-week period, ani­
animal” and CPCSEA (711/02/a/CPCSEA/2013).
mals in groups 2–5 were declared diabetic with hyperglycemia (blood
glucose level >200–300 mg/dl). The blood glucose level of selected
2.8.2. Acute toxicity study
diabetic animals was determined for further experimental procedure
According to the OECD Guideline (2021), an acute toxicity study was
(Table 3).
performed. The adult albino Swiss mice 20–30 g was allocated into four
groups containing three animals in every group. Overnight fasted ani­
2.8.3.2. Oral glucose tolerance test (OGTT). After overnight fasting, 0-
mals were administered orally with the PH formulation at various dose

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S. Majhi et al. Phytomedicine Plus 2 (2022) 100337

Fig. 4. Effect of PH on STZ induced Diabetic rats (Antihyperglycemic effect)

Results are displayed as mean ± SEM (n = 6 rats/group).
#
P<0.01; when Diabetic control compared with Normal control (unpaired t-test).
a
P<0.01; when STD, PH I (200 & 400 mg/kg) compared with respective “0′′ day BGL (1-way ANOVA, followed by Post Tukey’s test) (NC–Normal Control; DC-
Diabetic Control; STD-Standard Glibenclamide; PH I Polyherbal formulation I).

Fig. 5. Effect of PH on Liver Glycogen

Content on STZ induced Diabetic rats
Results are displayed as mean ± SEM (n
= 6 rats/group).
#
P<0.01; when Diabetic control
compared with Normal control (unpaired
t-test).
a
P<0.01; when PH I (200 mg/kg)
compared with Diabetic control (1-way
ANOVA, followed by Post Tukey’s test).
b
P < 0.001 when STD, PH I (400 mg/kg)
compared with Diabetic control (1-way
ANOVA, followed by Post Tukey’s test)
(NC–Normal Control; DC-Diabetic Con­
trol; STD-Standard Glibenclamide; PH I
Polyherbal formulation I).

min blood sample was taken from different groups of rats, namely,
normal, diabetic control, diabetic + PH extract 200 and 400 mg/kg, and
diabetic + glibenclamide 600 μg/kg by the retro-orbital sinus puncture,
and without any set back, a glucose solution (2 g/ml per kg) was
dispensed by gavage. Four more blood samples were withdrawn at 30,
60, 90, and 120 min after glucose administration. All the samples were
collected for the estimation of glucose using commercially available kits.
2.8.3.3. Hypoglycemic activity. Overnight fasted adult Wistar rats
(water was allowed ad libitum) were segregated into four different
groups containing 6 rats each. PH extract was administered orally in
dose of 200 and 400 mg/kg body weight. Glibenclamide (600 μg/kg)
was administered to one group, and 1 ml/kg body weight of 0.5% so­
dium CMC was administered to the control group. Commercially avail­
able kits were used to measure blood glucose level by glucose oxidase-
peroxidase method at different time intervals, namely, 0, 30, 60, 120,
and 240 min by retro-orbital plexus puncture.
2.8.3.4. Antihyperglycemic activity. At the end of the 28th day, the rats
were fasted for 16 h and blood was collected by retro-orbital plexus
puncture. The serum was separated by centrifugation and used to
measure blood glucose level.

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S. Majhi et al. Phytomedicine Plus 2 (2022) 100337

Fig. 6. Effect of PH on Lipid Profile of STZ induce Diabetic rats

Results are displayed as mean ± SEM (n = 6 rats/group).
@
P<0.01; when Diabetic control compared with Normal control (unpaired t-test).
#
P < 0.001; when Diabetic control compared with Normal control (Unpaired t-test).
a
P<0.01; when STD, PH I (400 mg/kg) compared with Diabetic control (1-way ANOVA, followed by Post Tukey’s test).
b
P < 0.001 when STD, PH I (200 & 400 mg/kg) compared with Diabetic control (1-way ANOVA, followed by Post Tukey’s test) (NC–Normal Control; DC-Diabetic
Control; STD-Standard Glibenclamide; PH I Polyherbal formulation I).

Fig. 7. Effect of PH on Liver Enzymes on STZ induced Diabetic rats

Results are displayed as mean ± SEM (n = 6 rats/group).
#
P < 0.01; when Diabetic control compared with Normal control (Unpaired t-test).
@
P<0.001; when STD, PH I (200 & 400 mg/kg) compared with Diabetic control (1-way ANOVA, followed by Post Tukey’s test) (NC–Normal Control; DC-Diabetic
Control; STD-Standard Glibenclamide; PH I Polyherbal formulation I).

2.8.4. Estimation of serum parameters retro-orbital plexus and collected in EDTA-coated tubes (Waynforth,
Blood glucose level was measured by GOD-POD method at every 7- 1980). Oral Glucose Tolerance Test (OGTT) and Hypoglycaemic activity
day interval. Blood was withdrawn using a glass capillary through were performed. On the 28th day, the rats were fasted overnight for

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S. Majhi et al. Phytomedicine Plus 2 (2022) 100337

Fig. 8. Effect of PH on Serum Creati­

nine on STZ induced Diabetic rats
Results are displayed as mean ± SEM
(n = 6 rats/group).
#
P < 0.01; when Diabetic group
compared with Normal control (Un­
paired t-test).
@
P<0.001; when STD, PH I (200 & 400
mg/kg) compared with Diabetic control
(1-way ANOVA, followed by Post
Tukey’s test) (NC–Normal Control; DC-
Diabetic Control; STD-Standard Gliben­
clamide; PH I Polyherbal formulation I).

Fig. 9. Effect of PH on Antioxidant

Parameters on STZ induced Diabetic
rats
Results are displayed as mean ± SEM
(n = 6 rats/group).
#
P < 0.01; when Diabetic control
compared with Normal control (Un­
paired t-test).
a
P<0.01; when STD, PH I (200 mg/
kg) compared with Diabetic control
(1-way ANOVA, followed by Post
Tukey’s test).
b
P<0.001; when STD, PH I (200 &
400 mg/kg) compared with Diabetic
control (1-way ANOVA, followed by
Post Tukey’s test) (NC–Normal Con­
trol; DC-Diabetic Control; STD-
Standard Glibenclamide; PH I Poly­
herbal formulation I).

blood glucose and biochemical parameters estimation. Collected blood
was centrifuged to obtain serum to measure antihyperglycemic activity
(Trinder, 1969). Further, the liver was collected from the sacrificed
animals and liver glycogen (Vries, 1954) was estimated.
2.8.5. Estimation of biochemical & antioxidant parameters
Serum lipid markers (Allain et al., 1974; Fossati and Prencipe, 1982;
Friedewald et al., 1972); Aspartate transaminase (AST or SGOT);
Alanine transaminase (ALT or SGPT) and creatinine level was measured
(Henry, 1974; Henry et al., 1960). Antioxidants like GSH- Reduced
glutathione (Ellman, 1959); Superoxide Dismutase (SOD) (Kono, 1978)
along with Protein (Lowry et al., 1951) and lipid peroxides (MDA)
(Ohkawa et al., 1979) were estimated using diagnostic kits.
2.8.6. Histopathological study of liver and kidney
The intact liver and kidney of STZ induced diabetic and non-diabetic
rats were dissected. The samples were kept in 10% formalin till further
process. The samples were sent for histopathological studies to deter­
mine any organ toxicity.
2.8.7. Statistical analysis
All the results were indicated in terms of mean ± SEM (n = 6 animals
per group). Statistical analysis was performed via unpaired student t-test
and one-way analysis of variance (ANOVA) followed by post-test of
Tukey’s. The significance was measured at P < 0.01 to 0.001.
3. Results
3.1. Polyherbal formulation (tablet) evaluation parameters
All the physicochemical parameters and trace metals were within
limits (Table 4) as per the Pharmacopeial specifications. Table 5 showed
that the physical properties of the granules, like bulk density, tapped
density angle of repose, Carr’s index, and Hausner’s ratio, were found to
be within limits, which indicates good flowability of the granules. The
polyherbal tablets were evaluated for various parameters such as color,
average weight, hardness, friability, and disintegration time, which
were found to be acceptable as per the pharmacopeial specifications.
Based on various specifications in Table 2, formulation Batch 9 was

8
S. Majhi et al. Phytomedicine Plus 2 (2022) 100337

Fig. 10. Histopathological studies of liver

Normal Control (a- intact central vein; b- radially arranged hepatocytes around central vein); Diabetic Control (c- apoptotic dark shrunken hepatocytes; D- enlarged
sinusoids with thick veins); Standard (glibenclamide- almost normal hepatic structure); PH I (200 mg/kg- radially arranged hepatocytes around central vein with
slight apoptosis); PH I (400 mg/kg- almost normal hepatic structure with radially arranged hepatocytes around central vein).

selected as the optimized batch. compared with diabetic group. Also, food (14.58%) and water (11.74%)
intake decreased remarkably (P < 0.01) in PH I (200 mg/kg) treated
3.2. Toxicity study categories in comparison to diabetic (STZ) rats.

Acute toxicity study of the PH I was nontoxic. Divergent doses of PH I
i.e., 200 mg/kg and 400 mg/kg were not showing any toxic or lethal
reaction at any point of the experimental period.
3.3. Body weight (BW), food and water intake
Before and after BW of the normal and diabetic group is shown in
Fig. 1. Significant decline in BW of diabetic group (P < 0.001) and
diabetic + PH I (200 mg/ kg) (P < 0.01) was observed. Administration of
glibenclamide and PH I (400 mg/kg) did not exhibit any remarkable (P
< 0.01) difference in BW in the diabetic group. The food and water taken
by all groups are displayed in Table 6. The diabetic (STZ) group revealed
a higher consumption of food (37.21%) and water (61.69%) as
compared to normal at P-value < 0.001. The diabetic group when
treated with glibenclamide and PH I (400 mg/kg), produced a remark­
able (P < 0.001) decrease in food (20.93% and 18.62%, respectively) as
well as the water intake (17.01% and 16.37%, respectively) when
3.4. Blood glucose level
3.4.1. OGTT
The values were obtained for the rats administered with 3 g/kg oral
glucose load following various treatments. Oral administration of
glucose has remarkably (P < 0.01) increased BGL as in diabetic control
at all time intervals as compared to the control group. Further, when the
diabetic control group was compared to any of the antidiabetic treat­
ments, the glucose level observed was remarkably less at all time points
for the latter (P < 0.001). The plasma glucose-lowering effects of the
treatment group was pronounced up to 90 mins. The comparative
decrease in BGL in the diabetic treatment group is as follows: PH I (200
mg/kg)> Glibenclamide > PH I (400 mg/kg) (Fig. 2).
3.4.2. Hypoglycaemic activity
The study unveils the hypoglycaemic effect of PH I in a normogly­
cemic group where fasting blood glucose levels remain unchanged in the

9
S. Majhi et al. Phytomedicine Plus 2 (2022) 100337

Fig. 11. Histopathological studies of Kidney

Normal Control (a- kidney tissue appeared normal with glomeruli); Diabetic Control (b- sclerosis of glomeruli, c-mononuclear cell infiltration, D- tubular degen­
eration); Standard (STD-glibenclamide); PH I (200 mg/kg- moderate glomerular necrosis and MNC infiltration); PH I (400 mg/kg- no abnormality in the architect).

control group over 4-h study period. Nevertheless, in groups adminis­
tered with glibenclamide and two different dose levels i.e., PH I (200 and
400 mg/kg), BGL was remarkably lower. The blood glucose reduction
was greatest 33.93% for glibenclamide (P < 0.001) followed by 33.84%
for PH I 400 mg/kg (P < 0.001) and 26.73% for PH I 200 mg/kg,
respectively. Thus, significant hypoglycaemic activity was observed for
all the treatment groups i.e., Glibenclamide, PH I 400 mg/kg, and PH I
200 mg/kg (Fig. 3).
3.4.3. Antihyperglycemic activity
The antihyperglycemic study revealed that fasting BGL was
remarkably 3-fold more (P < 0.01) in the STZ-induced group than that of
the control (hyperglycemia continued in diabetic control over a 4-week
period). The BGL observed in treatment groups (glibenclamide and PH I
200 and 400 mg/kg) was significantly lower. At end of the study period
(week 4), blood glucose reduction was greatest 66.42% for PH I (400
mg/kg) followed by 61.13% for glibenclamide and 57.48% for 200 mg/
kg PH I. PH I (200 mg/kg and 400 mg/kg) remarkably (P < 0.01)
showed dose-dependent blood glucose-lowering effect at termination
(Fig. 4).
3.5. Liver glycogen
The liver glycogen load significantly raised (1.2–1.8-fold) in treated
diabetic groups, i.e., 1.6-fold for glibenclamide (P < 0.001), 1.3-fold for
PH I (200 mg/kg) (P < 0.01), and 1.7-fold for PH I (400 mg/kg) (P <
0.001)-treated groups respectively, as compared to their diabetic con­
trol. Significant decline in liver glycogen level (P < 0.01) was observed
in diabetic control as compared to normal group (Fig. 5).

10
S. Majhi et al.
3.6. Estimation of biochemical parameters
3.6.1. Serum lipid profile
Administering PH I (200 & 400 mg/kg) revealed remarkable
decrease (P < 0.001) invariant lipid level as compared to diabetic con­
trol. HDL increased significantly (P < 0.01) in glibenclamide and PH I
400 mg/kg dose when compared to diabetic group. PH I (400 mg/kg)
was comparatively more effective than 200 mg/kg in declining TC, TG,
LDL, and VLDL & increasing HDL cholesterol compared to the diabetic
control group (Fig. 6).
3.6.2. Hepatic enzymes
As compared to the control group remarkable rise in SGOT, SGPT
activity (≅ 3.67 fold and ≅ 1.72 fold respectively) was obtained (P <
0.01) in the diabetic group. Treatment with PH I (200 & 400 mg/kg)
showed the recovery of parameters. A comparative and remarkable (P <
0.001) difference was observed in enzymes level between groups
(Fig. 7).
3.6.3. Serum creatinine
The creatinine content (≅ 1.21 fold) in the diabetic group was
remarkably increased (P < 0.01) as compared to control. Creatinine
content declined remarkably (P <0.001) in PH I 400 mg/kg, followed by
STD and PH I 200 mg/kg groups (Fig. 8).
3.7. Estimation of antioxidant parameters - GSH, SOD and MDA
The SOD and reduced glutathione content significantly declined (P
< 0.01) in the diabetic group and the MDA amount surged remarkably
(P < 0.01). STD and PH I (400 mg/kg) receiving groups (P < 0.001) and
PH I (200 mg/kg) receiving group (P < 0.01) have shown remarkable
rise in SOD respectively. Similarly, reduced glutathione increased
significantly for Glibenclamide (P < 0.01) and PH I 400 mg/kg (P <
0.001) receiving groups. A significant (P < 0.001) reduction in MDA
level was noticed for all the treatment groups. The overall result con­
cludes that a high level of SOD, reduced glutathione and low level of
MDA in PH I (400 mg/kg) is better than PH I (200 mg/kg) dose in the
diabetic group (Fig. 9).
3.8. Histopathological study of liver and kidney
Control animal’s liver exhibited normal structures consisting of
central vein, hepatocytes, separated from each other by blood sinusoids.
In the diabetic group, hepatic cells were misarranged with necrotic
areas. Enlarged sinusoids with thick veins were observed. Polyherbal
formulation (pH I) or glibenclamide treatments prevented these alter­
ations as observed by lesser signs of necrosis, inflammatory portal areas
visible (Fig. 10). The renal tissue of the control group showed a normal
appearance. Diabetic control rats showed sclerosis of glomeruli, tubular
degeneration, mononuclear cell (MNC) infiltration, and thickening of
the glomerular basement. PH I (200 mg/kg) administration showed
moderate glomerular necrosis and MNC infiltration. However, 28-day
treatment with glibenclamide and PH I (400 mg/kg) showed no ab­
normality and the architect of kidney tissue appeared normal (Fig. 11).
4. Discussion
The present script discusses the hypoglycaemic and antidiabetic ac­
tivity of Polyherbal formulation on normal and STZ-induced diabetic
animals. It also considers the antioxidant, antihyperlipidemic activity,
serum creatinine, hepatic enzyme markers of the extract at varying dose
levels (200 & 400 mg/kg). The current protocol proposes that the
chemical constituents in the polyherbal formulation such as alkaloids,
flavonoids, phenolics, triterpenoids, xanthones possess potent hypo­
glycemic, antihyperglycemic and antioxidant activity along with anti­
hyperlipidemic activity.
11
Phytomedicine Plus 2 (2022) 100337
STZ is a nitrosourea cytotoxic compound obtained from Streptomyces
achromogenes. It produces ROS which enters β-cells via GLUT and in­
duces breakdown of DNA, resulting in the deficiency of endogenous
insulin (Irudayaraj et al., 2012). The breakage of DNA is due to nitrourea
moiety. Changes usually start after 2hours of STZ administration, further
developing hyperglycemia and concomitant thrust in insulin concen­
tration (Lenzen, 2008). In the end, severe hyperglycemia is produced
with a decline in insulin level (Arulmozhi et al., 2010). The toxicity
studies revealed that there were no fatality or noxious reactions found in
the groups receiving variant doses of extract till the culmination of the
experimental period. Thus, the PH formulation was found to be safe as it
contains a variety of herbs that showcases a chain of phytoconstituents
showing efficacious results for the current protocol.
OGTT is a process to evaluate altered carbohydrate metabolism after
administering a glucose load. PH formulation was found to be effica­
cious in controlling fasting BGL, decreases hyperglycemia due to OGTT,
and suggests that rats served with non-identical doses of PH extract have
better glucose utilization ability (Ceriello, 2005). The results elaborate
that an improved level of glucose tolerance is because of insulin release
from β -cells. Also, improved glucose transportation and consumption
are observed in different doses of extract-treated groups (Santiagu et al.,
2012). For 28 days, oral administration of PH formulation resulted in
significant hypoglycaemic activity as compared to glibenclamide
(standard drug). This result suggested that the mode of action of PH
formulation and glibenclamide may be the same (Sharma and Kumar,
2011; Eidi et al., 2005; Hosseinzaded et al., 2002).
Glycogen displays a salient feature for the dumping of glucose in the
guise of intracellular energy source (stimulates glycogen synthesis &
inhibits glycogen phosphorylase). The stores of liver glycogen were
markedly depleted in diabetic rats. This can directly cause insulin
deficiency (Chandramohan et al., 2008). STZ-induced group treated
with PH formulation (200 and 400 mg/kg) ameliorates the liver
glycogen level closer to normal, which shows an increase in insulin
secretion level. Hence, a rise in the uptake and utilization of glucose may
be a possible mechanism for this observation (Vetrichelvan et al., 2002).
Insulin poses a prime action for the metabolism of carbohydrates and
lipids. It declines free fatty acids and lipase activity in adipose tissue
(Loci et al., 1994). Diabetes increases β-oxidation of fatty acids leads to
lipid peroxidation (Oberley, 1988) followed by generation of ROS.
Streptozotocin-induced diabetes increase TC and TG involved in heart
ailments (Ananthan et al., 2003). The herbal extracts showed effects due
to increase in glucose uptake, and decrease in FFAs mobilization (from
fat deposits). The outcome of the current protocol proposes that variant
doses of the formulation remarkably rectified the lipid abnormalities.
Increased lipid peroxidation impairs membrane fluidity and enzyme
activity. Thus, the products formed are detrimental to the body cells
(Baynes, 1995). PH can inhibit the process of damage due to oxidative
stress. This occurs due to certain antiperoxidative moieties that can
inhibit the process of lipid peroxidation. Endogenous glutathione is a
free radical scavenger for several intracellular enzymes and also a
co-substrate. It displays an important part in hydrogen peroxide
breakdown which needs to undergo oxidation utilizing reduced (GSH) to
oxidation form (GSSG) (Meister and Anderson, 1983). The GSH: GSSG
ratio shows the mechanism of cellular redox equilibrium in which
declined GSH is indicated in DM. A major reduction in the amount of
GSH and the ratio of GSH / GSSG in diabetes suggests an incapacitated
system of glutathione defense. In diabetes-induced animals rising
amounts of MDA could also contribute to the altered concentration of
glutathione (Maritim et al., 2003). Our findings suggested that GSH has
increased in the pH; implying its positive impact.
In mitochondria, glucose level overload can rotate the transport
chain of electrons; thereby creating an abundance of superoxide radicals
(Wiernsperger, 2003). In STZ diabetic treated rats, high blood glucose
can enhance the amount of radical superoxide, in addition to greater
SOD activity. Enhanced activity of SOD enhances H2O2 turnover because
SOD transforms superoxide radicals to hydrogen peroxide (H2O2),
S. Majhi et al.
(Kono and Fridovich, 1982). A specific dose of PH substantially
improved the amount of antioxidative defense SOD and avoids mem­
brane degradation by reducing lipid peroxidation as opposed to diabetic
regulation.
A lot of herbs have been already identified as hepatoprotective in the
formulation. The PH greatly lowered the elevated levels of liver en­
zymes. In general, kidney damage is associated with an increase in the
amount of plasma serum creatinine found in diabetes-induced in­
dividuals (Bartels and Boehmer, 1971), which declined after treatment
with pH. This might be due to its anti-hyperglycemic action by boosting
the tissue’s uptake and use of glucose.
The liver histopathology analysis showed that the diabetic group
STZ-induced has significantly damaged liver with cytotoxic damage that
shows necrosis and fibrotic modifications. In addition, glibenclamide
and PH healed, appearing defects with limited signs of hepatotoxicity as
a remedy. Kidney histopathology analysis showed that the control group
and the regular treated group have similar glomeruli and tubules
compared to the group treated with streptozotocin (representing ne­
crosis in the kidney). Low dose polyherbal and high dose treated group
showed near return of usual glomeruli and tubules formation.
As per microscopic analyses, the dispensing of glibenclamide and PH
greatly decreased pathological alterations caused by streptozotocin.
While glibenclamide showed good efficacy for various biochemical
variables, the study concludes that the most successful one was poly­
herbal formulations. In the 28-day course of the study, no toxicity was
observed for the liver and kidney. Thus, the polyherbal formulation can
be a perfect replacement for the prevalent hypoglycaemic medications
with a supplementary benefit of antihyperlipidemic behavior thereby
reducing the parallel potential risks for DM. For this purpose, herbal
formulations need potential provisions for clinical trials to be effective
in the treatment of diabetes mellitus and its role in the antidiabetic drug
trade.
5. Conclusion
The treatment for diabetic mellitus continues throughout life.
Therefore, soothing agents are cherished with little or no side effects.
One such perspective is the use of an alternative and complementary
medication system consisting of natural supplements. The study offers
proof of the efficacy of multiple plant extracts as a blended formulation
in regulating BGL as opposed to individual extract. The research offers
proof of the efficacy of multiple plant extracts as a polyherbal mixture in
regulating blood sugar as opposed to individual plant extract. Future
studies in human volunteers are required to assess if the above findings
can be adequately extrapolated to humans. It was a deliberate effort to
use natural remedies together with allopathic medications to reduce
long-term usage risk factors similar to DM and the health risks of the
allopathic procedure. This brings us a renewed glimmer of hope for oral
hypoglycaemic drugs of a new generation and could have favorable
impacts on T2DM.
Conflict of interest
The authors declare no conflict of interest.
Funding
The research was self-funded.
CRediT authorship contribution statement
Sagarika Majhi: Conceptualization, Methodology, Software. Lub­
han Singh: Methodology, Software. Madhu Verma: Visualization,
Investigation. Iti Chauhan: Formal analysis, Visualization. Raj kumari:
Visualization. Meenakshi Sharma: Visualization.
12
Phytomedicine Plus 2 (2022) 100337
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
Authors thank the National Bureau of Plant Genetic Research, New
Delhi and Department of Botany, Chaudhary Charan Singh University,
Meerut for the authentication of plant species. The authors also thank
the management of I.T.S College of Pharmacy & M.I.E.T, Meerut to carry
out the research process with all the help required.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.phyplu.2022.100337.
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