International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:13 No:04

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In vitro Antileishmanial, Cytotoxic and Antioxidant activities of Salvia bucharica leaves extract and its fractions.
Afroz R.Khan, Muhammad Javed Khan
Abstract Balochistan, province of Pakistan is blessed with the
treasure of numerous medicinal plants. Salvia bucharica has widely been used in folk medicine for its medicinal properties. The present study has been focused on the crude methanolic leaves extract (CME) and its fractions: chloroform fraction (CCF), acetone fraction (CAE) and aqueous fraction (AQF).The plant was investigated for its aniti leishmanial, cytotoxic and antioxidant activities. CME showed significant antileishmanial activity with IC50 value 72.31. The results of CCF and CAF showed maximum brain shrimp cytotoxic activity with ED50 values 2.76 g/ml and 1.68 g/ml respectively. The antioxidant activity of Salvia bucharica ranged from 31.98 to 42.1 g/ml. CCF showed maximum antoxidant activity with IC50 value 42.1 g/ml. The extract and its fractions were also appreciating for further biological studies in future.

easily accessible and inexpensive [12]. Leishmaniasis is considered one of the severe tropical diseases in numerous countries. It is caused by protozoa, Leishmania. Three major clinical manifestation of leishmaniasis are recognized: visceral, cutaneous and Muco-cutaneous leishmaniasis [13]. In developed countries, increasing number of AIDS cases has brought increase attention towards these diseases [14]. The main aim and objective of current study is to evaluate the antileishmanial, cytotoxic, and antioxidant activities of leaves and stem of Salvia bucharica, as no work has been reported yet. Furthermore, we are also presenting the antileishmanial results of Salvia bucharica in our study, as this plant has not been reported as antileishmanial agent. II. Plant material: MATERIALS AND METHODS

Index Term Antileishmanial, Cytotoxicity (Brine shrimp),

Antioxidant, Salvia bucharica leaves.

I. INTRODUCTION The plant kingdom is a treasure house of potential drugs and there has been an increasing awareness about their importance of medicinal plants [1]. They are used locally in the treatment of infections caused by fungi, bacteria, viruses and parasites. Different plants have been used as a source of inspiration in the development of novel drug [2]. Many plants species have been evaluated for their antimicrobial activity in the past twenty years and since then effectiveness of many medicinal plants in the cure of many diseases have been put to test in various laboratories [3,4]. Salvia genus is distributed in the temperate and tropical region and it is the largest genus of aromatic and ornamental herbs and shrubs [5]. Salvia belongs to the family of Lamiaceae, previosly called Labiaae. About 800 species were found throught the world. Central Asia and South-west Asia are significant centers for this very distinct genus. In Pakistan sixteen species of this genus are present [6]. In different parts of the world numerous species of genus Salvia are used as folk medicins [6]. For essential oils, numerous members of this family have been studied [7,8] . The antifeedant, antifungal activity has been shown from a variety of isolated bi-and-tricyclic diterpenes from the species of Salvia [9] . Decoction of S.bucharica has been traditionally used as a folk medicin for the cure of appendicitis and flowers are used for the treatment of throat complaint [10]. Lamiaceae, to which Salvia belongs, is famous due to its members having chemical constituents with anti-tumor activity [11]. Plants derived medicines are extensively used because they are comparatively safer than the synthetic alternatives, they are

Salvia bucharica leaves were collected from District Quetta, Balochistan province, Pakistan. Extraction and Fractionation: Fresh leaves were washed, sliced and dried under shade for 20 days at room temperature. The dried material was ground to fine powder using a mechanical grinder (IKAMF 10 basic micro fine grinder drive). The leave extract was prepared in analytical grade methanol (800 g in 4L) for 72 hours. Then the methanol was removed and residue was immersed in methanol for further seven days. Thereafter, the methanol was decanted and filtered with Whatman filter paper No 1. The filtrate was subsequently concentrated under reduced pressure at 45 C in rotatory evaporator (Stuart RE 300) and dried to constant weight (460 g) in vacuum oven (LINN high therm) at 45C. This was crude methanolic leave extract (CME). The CME was than further fractionized, where 100g of CME was suspended in 350 ml of distilled water. This aqueous suspension was further subjected to solvent-solvent extraction for four fractions, namely, (CCF) chloroform fraction, Acetone fraction (CAF) and Aqueous fraction (AQF). Biological activities: Following biological activities were performed on the extract and its fractions.

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International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:13 No:04 Antileishmanial assay: Culture of parasites: L. major promastigotes were isolated from a patient with Cutaneous leishmaniasis from (Bolan Medical complex), Quetta, Pakistan. The promastigotes were grown in NNN medium and then cultured in 199 medium supplemented with 10% fetal bovine serum x (FBS) (PAA laboratories Gmbh). Samples preparation: 25, 50, 250 and 500g/ml concentrations of CME and its fractions were prepared for invitro studies. The extracts were dissolved in DMSO and diluted in 199 medium containing 10% F.B.S. The final volume was adjusted to 2000 l with 199 medium, for each well a 24 well micro plate in all experiments. The final concentration of DMSO was 0.5% (v/v) as this concentration will not affect the parasite growth rate, mobility morphology [15]. 100 L. major parasites were transformed into each well, after hemocytometer counting, promastigotes were suspended to yield 1x106 cell/ml in each well, as reference drug. Amphotericin B was prepared in sterile DMSO at 20 g/ml concentration. The highest concentration of DMSO and 199 medium were also used for control groups. Micro plates were incubated at 24 C. The numbers of parasites were counted with a hemocytometer under a high microscope after 6, 12, 24, 48 hours. All the invitro experiments were run in triplicate and the results were expressed as a % inhibition in parasite numbers. The drug concentration required for 50% inhibition invitro (IC50) was calculated with parametric statistical procedure (Finney probitic analysis program) with the associated with 95% confidence interval [16]. Brine shrimp Cytotoxicity assay: The brine shrimp Cytotoxicity assay was performed by using the methodology according to the procedure described by [17]. Brine shrimp (Artemia salina) larvae used as test organisms, were hatched at 37 C in artificial sea water. Different concentrations i.e. 1000, 100, and 10 g/ml (control) of CME, CCF, ACF and AQF were in methanol and used against brine shrimp larvae. The death rate of these larvae was observed against all concentration of different fractions. For this purpose, 0.5ml sample of each and every fraction was taken in 20ml vial, solvent from each vial was evaporated followed by addition of 2ml of artificial sea water, 30 shrimps were transferred into each vial, final volume was adjusted to 5ml by artificial sea water and kept under florescence light at
0

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25 C for 24 hours. Test was performed in triplicate after this, deaths were counted, and percentage survival was counted with ED50 values were determined by (Finney Computer program). Antioxidant assay: The free radical scavenging activity was measured by using 2, 2-diphenyl-1-picryl-hydrzyl (DPPH) assay. DPPH radical assay radical assay was performed according to the procedure described by [18]. DPPH solution was prepared by dissolving 3.2mg in 100ml of 82% of methanol. A volume of 2800l of DPPH solution was added to glass vials followed by addition of 200 l of CME, leading to the final concentration of 100, 50, 25, 10 and 5 g/ml (negative control), mixture were shaken well and incubated in dark at 25C for 1 hour. Absorbance was measured at 517nm using spectrophometer (Pharma Spec 1700 Shimadzu). Ascorbic acid (AsA) was used as positive control. Each test was measured according to formula and IC50 were calculated by graphical method. Same procedure was then repeated with other fractions such as (CCF), (ACF) and (AQF). (%) scavenging effect = [(AC-AS)/ AS] x 100 Where; AC is the absorbance of negative control and AS is the absorbance of Test Sample. III. RESULTS AND DISCUSSION Crude Methanolic Extract (CME) of Salvia bucharica leaves was prepared and partitioned into three fractions i.e. CCF, ACF, and AQF. The plant crude extract their partitions were evaluated for their biological activities Antileishmanial, Brine shrimp Cytotoxicity and Antioxidant activities. Antileishmanial Activity We checked invitro antileishmanial effect of Salvia bucharica leaves extract and its fracions.The extract showed good inhibition activity against the promastigotes of L. major even with a concentration of 250 g/ml. (Table I) shows the IC 50 of extract and fractions ranged between 30.51 to 72.31 g/ml. CME was found to be more active than fractions. CME showed the highest antileishmanial activity of 72.31 % at 500 g/ml. The CAF was the weakest one showing IC50 >100 g/ml. The AQF showed good activity 55 % inhibition at 25 g/ml with IC50 30.51. DMSO and 199 culture controls were found to be inactive in all experiments. The reference drug Amphotercin B. was found to have 100% inhibitions

after 48 hours.

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International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:13 No:04

TABLE I In-vitro efficacy of Salvia bucharica leaves extract and its fractions.% Inhibition of death L. major parasite at different doses (g/ml).

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Extracts/ Fraction CME

Doses (g/ml) 25 50 250 500 25 50 250 500 25 50 250 500 25 50 250 500 25 50 250 500

Number of Promastigotes (1x 104) 56 60 41 25 65 69 55 40 81 75 66 65 45 52 48 25 100 100 100 100

% inhibition 44 40

(IC50)g/ml

72.31 59 75 33 36 45 60 10 12 >100 15 17 55 48 30.51 52 75 activities in Plant Extracts [19]. In present study, the ED50 values of Salvia bucharica leaves ranged from 1.03 to 2.76, while CCF showed significant activity with ED50 value of 2.76 g/ml. In contrast Fractions CME and CAF showed good activities with ED50 values of 1.03 and 1.68 g/ml respectively. AQF showed the lowest activity with ED50 value of >100 g/ml comparatively with Standard drug. 50.51

CCF

CAF

AQF

DMSO(ve)

Cytotoxic Activity Brine shrimp cytotoxity assay has been considered as prescribing assay for anti-fungal, anti-parasitological, antimicrobial and insecticidal activities. Brine shrimp assay in recommended to be a suitable probe for the pharmacological

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International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:13 No:04

TABLE II Cytotoxic activity of Salvia bucharica leaves extract and its fractions.

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Extract/ Fractions CME CCF CAF AQF DMSO(-ve) Etoposid (+ve)

Number of brine shrimp 30 30 30 30 30 30

% death at doses 1000g/ml 25 26 28 25 30 100g/ml 21 23 24 23 27 10g/ml 19 18 20 22 24

ED50 g/ml 1.03 2.76 1.68 >100 0.56

Antioxidant activity DPPH free radical scavenging assay was used to evaluate antioxidant potential of our samples. CME as well as its fractions showed effective free radical scavenging activity as determined by DPPH assay. The results of free radical scavenging are given in (Table III). The antioxidant activities ranged from 31.98 to 42.1 g/ml. CCF has showed maximum

antioxidant activity with the IC50 value of 42.1g/ml. On the other hand CME showed good antioxidant activity with IC50 value of 31.98 g/ml. While ACF and AQF showed lowest free radical scavenging activity and have IC 50 >100 g/ml. CCF has excellent free radical scavenging to Ascorbic acid. Salvia bucharica leaves extract and its fractions have outstanding pharmacological importance and it should be investigated further for Isolation, Purification and Characterization of precious compounds.

Table III DPPH scavenging antioxidant activities of CME and its Fractions of Salvia bucharica leaves extract.

Extract/ Fractions CME CCF ACF AQF ASA

100g/ml 80.12 73.89 55.32 27.12 95.05

50g/ml 74.69 58.30 40.44 13.32 94.81

25g/ml 40.24 33.71 10.28 90.01

10g/ml 20.10 20.10 86.3

5g/ml 9.46 9.46 44.7

REFERENCES

IC50 g/ml 31.98 42.1 >100 >100 5.4

IV. CONCLUSION The present study concluded that the crude methanolic extract of Salvia bucharica leaves exhibited significant antileishmanial activity. Furthermore, crude chloroform fraction showed maximum antoxidant activity. So, from the present study it can be concluded that more work can be done regarding the characterisation of active groups present in Salvia bucharica.

ACKNOWLEDGEMENT We are thankful to the Higher Education Commission of Pakistan and University of Balochistan for financial support. We thank Prof. Dr. Mudassir.Asrar.Zaidi Botany Department University of Balochistan, Quetta, Pakistan for identifying the plant.

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