BIOLOGICAL STUDIES ON LEAVES AND STEMS OF BEGONIA PICTA

BIOLOGICAL STUDIES ON LEAVES AND STEMS OF BEGONIA PICTA

Shrestha Nisha^*1, Khanal Dharma Prasad ² and Itani Renuka ¹

National Model College for Advance Learning ¹, Tribhuvan University, Kathmandu, Nepal.

Department of Pharmacy ², Manmohan Memorial Institute of Health Sciences, Tribhuvan University, Kathmandu, Nepal.

ABSTRACT: Aim / Background: Aim of this study is to provide scientific evidences that supports the traditional use of Begonia picta; helps to prepare Begonia picta Ayurvedic and / or allopathic formulation for immediate use of herb. Methods: In present study biological screenings were done by following Cytotoxicity pre-screening by Brine shrimp lethality bioassay, anti-hyperglycemic activity evaluation by oral glucose tolerance test and anti-inflammatory effect evaluation by hind paw edema method. Results: The brine shrimp lethality bioassay showed that the hexane and methanolic extracts of Begonia picta was mildly toxic to Artemia salina naupalii having LC₅₀ values 162.0690 µg/ml and 237.263 µg/ml respectively. The highest percentage of blood glucose lowering effect was found in 180 minutes for standard drug (32.52%), Begonia picta extract 200 mg/kg (39.62%), and 400 mg/kg (43.17%). Anti-inflammatory activity test result showed that maximum inhibition activity observed during 5 hour: 31.74% of standard, 28.08% of test (200 mg/kg) and 29.78% of test (400 mg/kg). Conclusion: The brine shrimp lethality bioassay confirmed that the hexane and methanolic extracts of Begonia picta was found to be mildly toxic. The results of anti-hyperglycemic activity test demonstrate that the methanolic extract possesses anti-hyperglycemic potential that was time and dose dependent. We can conclude the Begonia picta extract has anti-inflammatory activity which was both time and dose dependent.

Begonia picta, Brine shrimp lethality bioassay, Anti-hyperglycemic activity, Anti-inflammatory activity

INTRODUCTION: Medicinal plants have been used since ancient times for the treatment of human ailments. Interest in medicinal plants has been shown throughout the world because of the safe and effective constituents of plant products. The increasing demand for herbal medicines, both in the developing and developed countries, has provided the stimulus for the workers in this field to maintain the quality and purity of their herbal raw materials and finished products.

The standardization problem relating to herbal drugs arises from the complex composition of drugs that are used in the form of whole plants, plant parts or extracts obtained there from. To ensure reproducible quality of any herbal remedy, proper control of starting material is utmost essential ¹.

Nepal occupying the central part of Himalayas is rich in flora and fauna with more than eight hundred species. These herbs have been integral part of traditional medicinal practices of indigenous community in Nepal. Traditional medicine that represents the main alternative method which has its basis on indigenous knowledge gained from ancestral experiences. This knowledge is scientifically undocumented and still communicated verbally ².

Begonia picta is native plant of Himalaya region of Nepal. Among the herbal plants of Nepal, Begonia picta is a small tuberous species from mountains Nepal ³. Traditionally Begonia picta (magarkannche) is used in pained nipple in the form of wild herb/ whole plant. The juice whole plant is taken to relieve headache and also consumed in treatment of peptic ulcer. The paste is applied to stop bleeding from cuts and wound and is applied externally on ringworm and scabies. The root juice is used as eyes wash to treat conjunctivitis ⁴. The whole plant is feed to sterile animals to help them conceive. Whole plant is used as appetizer and juice of leaves about 4 teaspoonfuls 3 times a day is given to relieve the fever ⁵. Plant decoction is used in colic and dyspepsia ⁶. Begonia picta was being eaten raw or as pickle in Sikles by Gurungs and adjoining areas of Pokhara. The leaves have an acidic taste and are eaten raw as well as cooked for its delicious, sour taste in Palpa. This result is also supported by similar findings of the species being consumed as ‘Chatni (kind of pickle)’ by the local people in Daman and Dolakha. Paste of young shoot is also taken for respiratory tract infections.

Begonia picta also called patherchattha in India is used in dysentery and mouth ulcer ⁷. Powder of dry tuber used in case of renal calculi ⁸. Root infusion is taken orally to cure constipation ⁹. Begonia whole plant is used in mumps ¹⁰. Anti-inflammatory activity test of Begonia picta will explore the traditional uses related to inflammation. Diabetes is considered a “modern day epidemic” and is rightly recognized as a global public health issue ¹¹. Diabetes Mellitus is a common metabolic disorder in which amount of serum glucose is increased and will cover 5.4% of population by year 2025 ¹². Trends in the last 10 years are influencing the supply and demand for healthcare in diabetes ¹³. The medicinal values of Begonia malabarica as antidiabetic agents had established according to published studies ¹⁴, but not of Begonia picta, which is commonly found and traditionally used herb of Nepal. The hypoglycemic activity test on Begonia picta will help to explore the use of Begonia picta in addition to the traditional use.

MATERIAL AND METHODS:

Study Design: Descriptive and experimental research designs were used in this research.

Plant Material: The plant materials: leaves and stems were collected from Godavari Kunda Samudayic Ban Upabhokta Samuha Ward No. 5 Lalitpur, Nepal. During June/July 2014 and was duly identified as Begonia picta in National Herbarium and Plant Laboratory, Godavari, Lalitpur.

Animal and Organism Taken: Swiss Albino rat, Artemia salina naupalii.

Animal Handling: Swiss Albino wistar rats of both sexes (1.6 to 2.2 gram) were obtained from Natural Product Resource Laboratory Thapathali Kathmandu, Nepal. They were kept, fed, cared and handled by following the ethical guidelines for the care and use of animals in health research in Nepal ¹⁵.

Chemical and Apparatus used: Chemicals used; hexane, ethyl acetate, methanol, Glimepiride (laboratory standard drug, Lomus Pharmaceuticals Pvt. Ltd., Indomethacin SR capsule 75 mg (SR Drug Laboratories Pvt. Ltd.,).

Apparatus Used: Soxhlet Apparatus, Electric grinder, Rotary shaker (Associated Scientific Technologies Delhi, India), Glucometer (Gluco Dr. auto. Allmedicus Co. Ltd.,), Syringe lifeline (Everest med Pvt. Ltd.,) Vernier caliper (Ralson India), Incubator.

Processing of Samples: Drying was followed by shade drying method. The dried plant materials were crushed into powder by electric blender and subjected to extraction by using Soxhlet apparatus. The extracts obtained were dried by using Rota vapour drier and solid extracts were preserved in refrigerator at 4 °C for biological screening experiments.

Brine Shrimp Lethality Bioassay: In order to study the toxicity of medicinal plant, brine shrimp bioassay which based on the ability to kill laboratory cultured brine shrimp Artemia salina naupalii and lethality is evaluated after 24 hour. The commercial availability of inexpensive brine shrimp eggs, low cost and ease of performing the assay make brine shrimp lethality assay is a very useful bench-to method ¹⁶. The brine shrimp assay was proposed by Michael et al and later developed by Vanhaecke et al., ¹⁷. The brine shrimp assay is very useful tool for the isolation of bioactive compound from plant extract. The cytotoxicity level would give an indication about the plant ability as an antitumor agent.

TABLE 1: CONDITION REQUIREMENT TO CULTURE ARTEMIA SALINA NAUPALII

Preparation of Sea Water: Artificial sea water was prepared by dissolving sodium chloride, potassium chloride, sodium bicarbonate, sodium sulphate, Boric acid, calcium chloride, magnesium chloride, sodium EDTA.

Sample Preparation: Sample was prepared by dissolving 250 mg of each crude drug extracts in 250 ml of seawater to give stock solution. DMSO was also added in the samples which were not fully dissolved. Thus the concentration of stock solution that was prepared is 10 mg/ml. Seven different concentrations namely 1000 µg/ml, 500 µg/ml, 250 µg/ml, 125 µg/ml, 75 µg/ml, 50 µg/ml, 25 µg/ml were prepared in triplicate by serial dilution from stock solution and each concentration was made up to 100 ml with the salty sea water. A control was prepared by using only DMSO and sea water.

Hatching of Shrimp: Brine shrimp eggs were hatched in a 1000 ml beaker filled with sea water which was kept inside the incubator. Two torches light were made clamed above the beaker for continuous illumination and oxygen pump was dipped inside the beaker for oxygen supply. The shrimp eggs were sprinkled into the sea water containing beaker. After 24 hour hatched nauplii were collected for assay.

Bio-assay: Ten larvas of brine shrimp (naupalii) were transferred to each of the prepared test tube of different concentration using a syringe. To facilitate easy transfer, naupalii were counted under a magnifying lens (3 xs). The vials were maintained under illumination. The shrimps were counted after 24 hours which are survived. The total death and percentage mortality (death) at each dose level and control were determined.

Counting of Naupalii: After 24 hour the numbers of survivors in each triplicate were counted against illuminated background and percentage of death at each level dose level and control was calculated using following formula:

% mortality = Number of death × 100 ⁄  Number of death + Number of death

Those naupalii which did not show any movement were considered dead.

TABLE 2: THE TOXICITY LEVELS OF EXTRACTS

Statistical Analysis: For each extracts or sample the lethal concentration that causes 50% death (LC₅₀) was calculated at 95% confidence interval by linear regression analysis. A regression line equation was derived for each extracts by trend line in MS Excel 2010, and LC₅₀ was calculated using the equation. Make a graph on organism mortality (in %) related to logarithm to concentration of tested compound. Using the values on died individuals in given concentration determines the presence of mortality according to this formula;

Mmct = NMN × 100 ⁄ NO

Where, Mmct = mortality of individual in time t (%); NMN= average number of died individuals; NO= initial number of living individuals put into every concentration at the test start assess the EC₂₅, EC₅₀, EC₇₅, EC₁₂₅, EC₂₅₀, EC₅₀₀, EC₁₀₀₀ values using non-linear regression where mortality is related to decimal logarithm of concentration ^{18 - 20}.

Anti-hyperglycemic Effect Evaluation:

Oral Glucose Tolerance Test: Oral glucose tolerance tests were carried out as per the procedure previously described by Joy and Kuttan (1999) ²¹, Faisel et al., ²² with some modification. Oral glucose tolerance test is simple method to measure body’s ability to use or remove excess sugar from blood and also used to screen hypoglycemic properties of products / extracts. Fasting greatly decrease the rate of disappearance of glucose from the blood after hyperglycemia, while in non-fasting condition, tolerance is very high. So, products / extracts are given to overnight fasted (16 - 18hr) rat orally or intraperitoneally to measure tolerance. Then, a load of glucose was given orally and their blood glucose level is determined. The tolerance of mice to glucose gives indication of presence of hypoglycemic activity.

Preparation of Standard Drug Solution: As Glimepiride human optimum dose per day is 6 mg/kg. For rat 0.43 mg/kg per oral was given, for this 1 mg / 2 ml of stock solution was prepared. From stock solution 0.2 ml, 0.22 ml and 0.23 ml solution were taken and given to 200 g, 220 g and 230 ml rat respectively.

Preparation of Plant Extract: Stock solution of 100 mg/ml was prepared by dissolving 0.1 g methanolic extract was taken and few drops of Tween 80 was added to mix it and dissolved in distilled water. Final volume was adjusted to 10 ml with distilled water. Further 0.4 ml, 0.44 ml, 0.34 ml, 0.4 ml, 0.88 ml and 0.84 ml were taken and given orally to 200 g, 220 g, 170 g, 200 g, 220 g and 210 g rat respectively.

Preparation of Glucose Solution: Glucose of 10 g was dissolved in 50 ml of distilled water. From this solution 3 g/ kg/ oral dose was given to group 2 to 4. For this 1.5 ml, 3.3 ml, 3.45 ml, 1.5 ml, 3.3 ml, 2.55 ml, 1.5 ml, 3.3 ml and 3.15 ml solutions were taken and given to rat having weight of 200 g, 220 g, 230 g, 200 g, 220 g, 170 g, 200 g, 220 g, and 210 g respectively.

Procedure: In this method 12 hour fasted, healthy rats were divided into 4 groups of 3 animals in each.

Blood glucose level was measured using Gluco Dr. auto Glucometer.

• Group 1 was served as control group that received 10 ml/kg of distilled water.
• Group 2 which received the standard drug Glimepiride (0.43 mg/kg) dissolved in distilled water.
• Group 3 (Test) which received methanol extract of 200mg/kg dissolved in distilled water.
• Group 4 (Test) which received methanol extract of 400 mg/kg dissolved in distilled water.

Initially blood glucose level of each rat was noted by using the Gluco. Dr. Glucometer. Then each rat of Group 1, 2, 3 and 4 was given respective sample orally as mentioned above. After 30 min: this treatment, glucose (3g / kg) was given orally through a feeding tube to the each animal of all groups. Blood was drawn from the tail vein of animals at an interval of 30 min; at 0 time, 30 min, 60 min and 120 min. The percentage lowering of blood glucose levels (BGL) were calculated by using following formula:

Percent lowering of blood glucose level = 1- W_e/W_c *100

Where W_e and W_c represents the blood glucose concentration in glimepiride or methanol extract administered rat (Groups 2, 3 and 4), and control rat (Group 1) respectively ^{23 - 25}.

Anti-inflammatory Activity Test:

Fresh Egg White Induced Paw Edema: Inflammation can be defined as a reaction of living cell or tissue to injury, infection. Inflammation is characterization by pain, swelling, redness and heat or fever. Inflammation is evoked by condition that bring about the release of inflammatory mediators such as histamines, prostaglandins, nitric oxide, serotonins, leucotrines, cytokines, platelet activating factors and substance P. Histamine and other mediators of inflammation increase vascular permeability at various times after injury. Chemically induced vascular permeability can causes an immediate reaction and its inhibitions suggests that the sub-cutaneous administration of test methanol extract of leaves of Begonia picta may effectively suppress the exudative phase of acute inflammation induced by undiluted fresh egg white ²⁶.

Preparation of Standard Drug (Indomethacin SR 75mg) Solution: Two indomethacin SR capsules were taken and its average weight was taken. Powder of each capsule was mixed. 0.2884 g powder was taken and was dissolved in 20 ml distilled water which contain 150 mg indomethacin. From the prepared solution 5 mg/ kg/ oral dose was given to standard group rat. For this 0.12 ml, 0.15 ml and 0.14 ml solutions were given to 200 g, 220 g and 210 g rat respectively.

Preparation of Plant Extract: Stock solution of 100 mg/ml was prepared by dissolving 0.1 g methanolic extract was taken and few drops of Tween 80 was added to mix it and dissolved in distilled water. Final volume was adjusted to 10 ml with distilled water. Further 0.4 ml, 0.44 ml, 0.34 ml, 0.4 ml, 0.88 ml and 0.84 ml were taken and given orally to 200 g, 220 g, 170 g, 200 g, 220 g and 210 g rat respectively.

Preparation of White Egg Albumin: One egg was brought from the market near the college at the day of test start. It was broken and white part was separated from yellow part of egg. White part was mixed in cleaned mixture to make it syringiable. From this 0.05 ml was taken and given to each rats of group 2 to 5 to induce paw edema.

Procedure: The anti-inflammatory test of methanol extract of Begoni picta was performed by following the method Arun et al., ²⁷ with some modifications. The Swiss albino rats were divided into five groups. Each consists of 3 animals.

Group 1: Served as negative control (received only distilled water 2 ml/ kg/ oral).

Group 2: The second group served as control (received indomethacin SR 5 mg/ kg/ oral).

Group 3: The third group served as test (received methanol extract of Begonia picta 200 mg/kg/oral).

Group 4: The fourth group served as test (received methanol extract of Begonia picta 400 mg/kg/oral).

Group 5: The fifth group served as positive control (receive only undiluted egg white in sub-planter region).

Edema was induced by administration of 0.05 ml of undiluted fresh egg white in the sub-plantar region of group 2, 3, 4 and 5. The paw volume was measured by marking the point on swell paw by permanent marker at 0 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours after the injection of undiluted fresh egg white using Vernier caliper. Then percentage inhibition of edema is calculated by using formula ²⁸.

Percentage inhibition of edema = [(mean of control – mean of test) / mean of control] ×100

Acute Toxicity Test: Acute toxicity of methanolic extract of Begonia picta extract was tested on Swiss albino mice following Organization for Economic Cooperation and Development guidelines. This test was performed in Natural Products Research Laboratory, Department of Plant Resource, Thapathali Kathmandu, Nepal. The mice were subjected to intraperitoneal injection of methanolic extract of Begonia picta (0.2 ml / 20 gm) and kept fasted for overnight providing only water. Then, the animals were given free access to food and water and observed for a period of 48 h. The numbers of death occurred during the period were noted ^{29 - 31}.

RESULTS AND DISCUSSION:

Brine Shrimp Bioassay:

FIG. 1: RESULT OF BRINE SHRIMP LETHALITY BIOASSAY

FIG. 2: LC₅₀ VALUE OF DIFFERENT EXTRACT OF BEGONIA PICTA

FIG. 3: PHOTO TAKEN DURING BRINE SHRIMP LETHALITY BIOASSAY TEST

Anti-hyperglycemic Activity Test: Glucose administration 3 gm/kg glucose cause rise in blood glucose level where as after treatment of 200 mg/kg, 400 mg/kg methanol extract of Begonia picta and 0.43 mg/kg. Glimiperide led to a dose dependent drop in blood glucose levels.

FIG. 4: ILLUSTRATION OF BLOOD GLUCOSE LEVEL (BGL) IN VARIOUS TIME PERIODS

FIG. 5: REPRESENTATION OF PERCENTAGE BGL LOWERING EFFECT OF STANDARD AND METHANOLIC EXTRACT OF BEGONIA PICTA

FIG. 6: PHOTO TAKEN DURING ANTI-HYPERGLYCEMIC ACTIVITY TEST 

Anti-inflammatory Activity Test: Edema was induced by administration of 0.05 ml of undiluted fresh egg white in the sub-plantar region of group 2, 3, 4 and 5. After standard anti-inflammatory drug and Begonia picta extract administration; paw edema was gradually decreased in dose dependent manner within different time intervals.

FIG. 7: ILLUSTRATION OF PAW DIAMETER IN VARIOUS TIME PERIODS

FIG. 8: PERCENTAGE INHIBITION OF PAW EDEMA BY STANDARD DRUG AND METHANOLIC EXTRACT OF BEGONIA PICTA

FIG. 9: ANTI-INFLAMMATORY TEST

Toxicity: No abnormal behaviour was seen in seven days observation.

DISCUSSION: The brine shrimp lethality bioassay for hexane and methanolic extracts of Begonia picta was found to be mildly toxic. The LC₅₀ of hexane and methanolic extract were 162.0690 µg/ml and 237.263 µg/ml respectively. The toxicity of ethyl acetate extract was found to be greater than that of hexane and methanolic extracts. The LC₅₀ of ethyl acetate extract of Begonia picta is not possible to calculate because LC₅₀ is predicted to be below the concentration of 25 µg/ml that the lowest concentration of this study. The anti-hyperglycemic effect evaluation of methanolic extract of Begonia picta (200 mg/kg/oral and 400 mg/kg/oral) doses administered in Swiss albino rat showed that the decreased in blood glucose level in 60, 120, and 180minutes during test. The oral hypoglycemic drug glimepiride (0.43 mg/kg/oral) was taken as standard drug to compare the anti-hyperglycemic effect of Begonia picta extract.

Glimepiride is an antidiabetic agent that lowers blood glucose in patients with type-2 diabetes by stimulating the release of insulin from functioning pancreatic beta cells. Glimepiride also produces an increase in the sensitivity of peripheral tissues to insulin via an extra pancreatic mechanism. At extract doses of 200 mg/kg extract the percentage blood glucose lowering were 17.149%, 32.26%, and 39.62% at 60, 120 and 180 minutes respectively. At extract dose 400 mg per kg body weight rat, the percent lowering of blood glucose by the extract were 27.35%, 31.72% and 43.17%: at 60, 120 and 180 minutes respectively. The standard drug glimepiride dose 0.43 mg/kg, the percentage lowering blood glucose was 17.94%, 26.83% and 32.52% at 60, 120 and 180 minutes respectively. The highest blood glucose lowering effect (32.52%) was found in 180 minutes for standard drug and extract; Begonia picta extract 200 mg/kg (39.62%) and 400 mg/kg (43.17%).

The anti-hyperglycemic activity of extract was found to be time and dose dependent where % BGL lowering effect of extract dose 400 mg/kg is greater than that of 200 mg/kg. The % BGL lowering effect of Begonia picta extracts at 180 minutes is greater than the standard drug. The standard drug Glimeperide has T_max 2 to 3 hours. The extract has greater % BGL lowering effect than glimepiride; it may be because the glimepiride was not completely dissolved in distilled water.

The anti-inflammatory activity evaluation of methanolic extract of Begonia picta (200 mg/kg/oral and 400 mg/kg/oral) doses administered in Swiss albino rat showed that the inflammation of paw was decreased from 1 hour to 6 hours. The non-steroid anti-inflammatory drug indomethacin (5 mg/kg/oral) dose was taken as standard drug to compare the anti-inflammatory activity of Begonia picta extract. Indomethacin is a non-steroidal anti-inflammatory at Drug (NSAID) with anti-inflammatory, analgesic and antipyretic activity. Its pharmacological effect is thought to be mediated through inhibition of the enzyme cyclooxygenase (COX), the enzyme responsible for catalyzes the rate-limiting step in prostaglandin synthesis via the arachidonic acid pathway.

The percentage inhibition of edema by methanolic extract of Begonia picta 200 mg/kg were 8.89, 16.92, 24.96, 23.41, 26.55, and 22.20% at 1, 2, 3, 4, 5, and 6 hours respectively. The percentage inhibition of edema by methanolic extract of Begonia picta 400 mg/kg were 10.72, 19.60, 23.18, 23.41, 28.02, and 24.23% at 1, 2, 3, 4, 5 and 6 hours respectively. The percentage inhibition of edema by standard drug 0.43 mg/kg were 14.209, 24.12, 28.54, 29.01, 31.81 and 29.31% at 1, 2, 3, 4, 5, and 6 hours respectively. The maximum inhibition activity was observed during 5 hr: 31.74% of standard 28.08% of test (200 mg/kg) and 29.78% of test (400 mg/kg). It is clear that the anti-inflammatory activity of extracts is time and dose dependent. It was dose dependent because % inhibition of edema at 5 hr of extract dose 400 mg/kg is greater than that of 200 mg/kg.

CONCLUSION: The brine shrimp lethality bioassay showed that the hexane and methanolic extracts of Begonia picta were mildly toxic to Artemia salina naupalii having LC₅₀ values 162.0690 µg/ml, and 237.263 µg/ml respectively. In oral glucose tolerance tests, conducted in glucose-loaded Swiss albino rats: methanolic extract of Begonia picta has potential to reduced blood glucose level. The results provide the information that the methanolic extract possesses antihyperglycemic activity. On the pharmaco-logical point of view Begonia picta appears to be a valuable plant, which can be useful in the therapy of diabetes. The Swiss albino rat whose paw was inflamed and was treated with standard drug (indomethacin) and Begonia picta extract. Level of inflammation in treated rat was compared with untreated control rat at constant time intervals of 0, 1, 2, 3, 4, 5, 6 and 24 hour.

The experiment result showed that the Begonia picta extract has potential anti-inflammatory activity. The anti-inflammatory activity may be due to the inhibition of release of histamine, serotonin and kinins after the injection of egg albumin, and this also retarded the release of prostaglandin-like substance. It will be better to identify the active compounds that have cytotoxic, anti-inflammatory and hypoglycemic activities of Begonia picta.

ACKNOWLEDGEMENT: On the occasion of presenting this study words seem insufficient to express my deep sense of gratitude, sincere and heartfelt thanks to Supervisor Dr. Dharma Prasad Khanal, faculty member of National Model College For Advance Learning for his excellent guidance, critical supervision, keen observation, continuous encouragement, and support. I gratefully acknowledge to the department of pharmacy, National Model College for Advance Learning for providing the required facilities to complete our research work.

CONFLICT OF INTEREST: The authors declared no conflicting interest.

REFERENCES:

1. Ganesan P, Kumar CS and Bhaskar N: Antioxidant properties of methanol extract and its solvent fractions obtained from selected Indian red seaweeds. Bioresource Technology 2008; 99: 2717-23.
2. Manandhar NP: Native phytotherapy among the Raute tribes of Dadeldhura district, Nepal. Journal of Ethnopharmacology 1998; 60: 199-206.
3. Rajbhandary S, Hughes M and Shrestha KK: Distribution patterns of Begonia species in the Nepal Himalaya. Botanica Orientalis: Journal of Plant Science 2010; 7:73-8.
4. Rajbhandary S: Traditional Uses of Begonia Species (Begoniacae) in Nepal. Journal of Natural History Museum 2015; 27: 25-34.
5. Malla B and Chhetri R: Ethnoveterinary practices of some plant species by ethnic people of Parbat district, Nepal. Kathmandu University Journal of science, engineering and technology 2012; 8: 44-50.
6. Bhat JA, Kumar M and Bussmann RW: Ecological status and traditional knowledge of medicinal plants in Kedarnath Wildlife Sanctuary of Garhwal Himalaya, India. Journal of Ethnobiology and Ethnomedicine 2013; 9: 1.
7. Joshi KR, Devkota HP, Nakamura T, Watanabe T and Yahara S: Chemical constituents and their DPPH radical scavenging activity of Nepalese crude drug Begonia picta. Records of Natural Products 2015; 9: 446.
8. Khajuria AK and Bisht N: Ethnomedicinal plants used to treat Nephrolithiasis: A case study Pauri (PAURI Garhwal), Uttarakhand. synthesis 2017; 2: 5.
9. Rana SK, Oli PS and Rana HK: Traditional botanical knowledge (TBK) on the use of medicinal plants in Sikles area, Nepal. Asian Journal of Plant Science and Research 2015; 5: 8-15.
10. Tamang R, Thakur C, Koirala D and Chapagain N: Ethno-medicinal Plants Used by Chepang Community in Nepal. Journal of Plant Resources 2017; 15: 21.
11. Tebbitt MC: Begonias: cultivation, identification and natural history. Portland, Or: Timber Press 271p ISBN 2005; 881927333.
12. Zarshenas MM, Khademian S and Moein M: Diabetes and related remedies in medieval Persian medicine. Indian journal of endocrinology and metabolism 2014; 18: 142.
13. Bottomley JM and Raymond FD: Pharmaco-economic issues for diabetes therapy. Best Practice and Research Clinical Endocrinology and Metabolism 2007; 21: 657-85.
14. Pandikumar P, Babu NP and Ignacimuthu S: Hypoglycemic and antihyperglycemic effect of Begonia malabarica in normal and streptozotocin induced diabetic rats. Journal of ethnopharmacology 2009; 124: 111-5.
15. Pahari SK, Singh SP, Banmali MP, Thaler FJL and Rathour MSS: ethical guidelines for the care and use of animals in health research in Nepal.
16. Mclaughlin JL, Rogers LL and Anderson JE: The use of biological assays to evaluate botanicals. Drug information journal 1998; 32: 513-24.
17. Vanhaecke P, Persoone G, Claus C and Sorgeloos P: Proposal for a short-term toxicity test with Artemia nauplii. Ecotoxicology and environmental safety 1981; 5: 382-7.
18. Musa AA: Cytotoxicity Activity and Phytochemical Screening of Cochlospermum tinctorium Perr Ex A. Rich Rhizome 2012.
19. Krimat S, Dob T, Toumi M, Kesouri A and Noasri A: Assessment of phytochemicals, antioxidant, antimicrobial and cytotoxic properties of Salvia chudaei et Trab. endemic medicinal plant from Algeria. J Mater Environ Sci 2015; 6: 70-8.
20. Naidu JR, Ismail R and Sasidharan S: Acute oral toxicity and brine shrimp lethality of methanol extract of Mentha Spicata (Lamiaceae). Tropical Journal of Pharmaceutical Research 2014; 13: 101-7.
21. Joy K and Kuttan R: Anti-diabetic activity of Picrorrhiza kurroa extract. Journal of Ethnopharmacology 1999; 67: 143-8.
22. Faisal M, Hossain AI, Rahman S, Jahan R and Rahmatullah M: A preliminary report on oral glucose tolerance and antinociceptive activity tests conducted with methanol extract of Xanthosoma violaceum aerial parts. BMC complementary and alternative medicine 2014; 14: 335.
23. Shaha SR, Rahmatullah M: Oral glucose tolerance and analgesic studies with methanol extract of Brassica alba seeds. World J Pharm Pharmaceut Sci 2015;4:207-15.
24. Hossain AI, Faisal M, Rahman S, Jahan R and Rahmatullah M: A preliminary evaluation of antihyperglycemic and analgesic activity of Alternanthera sessilis aerial parts. BMC complementary and alternative medicine 2014; 14: 169.
25. Rahman S, Jahan R and Rahmatullah M: Effect of paddy husk extracts on glucose tolerance in glucose-induced hyperglycemic mice. World J Pharm Pharmaceut Sci 2014; 3: 111-20.
26. Payne DN: Nitric oxide in allergic airway inflammation. Current opinion in allergy and clinical immunology 2003;3:133-7.
27. Arun C, Kumar RS, Srinu S, Babu GL, Kumar GR and Babu JA: Anti-inflammatory activity of aqueous extract of leaves of Solena amplexicaulis. Intl J Res Pharma Biomed Sci 2011;2:1617-9.
28. Okoye NN, Ajaghaku DL, Okeke HN, Ilodigwe EE, Nworu CS and Okoye FBC: beta-Amyrin and alpha-amyrin acetate isolated from the stem bark of Alstonia boonei display profound anti-inflammatory activity. Pharmaceutical biology 2014; 52: 1478-86.
29. Co-operation of Development. Test No. 423: Acute Oral Toxicity-Acute Toxic Class Method: OECD Publishing 2002.
30. Sari LM, Utami S, Chairul C, Subita G, Whulandhary Y and Auerkauri E: Acute oral toxicity study of Areca catechu Aqueous extract in sprague-dawley rats. Asian J Pharm Clin Res 2014; 7: 20-2.
31. Yuan G, Dai S, Yin Z et al.: Toxicological assessment of combined lead and cadmium: acute and sub-chronic toxicity study in rats. Food and chemical toxicology 2014; 65: 260-8.
    

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    How to cite this article:

    Nisha S, Prasad KD and Renuka I: Biological studies on leaves and stems of Begonia picta. Int J Pharm Sci Res 2018; 9(3): 1167-75.doi: 10.13040/IJPSR.0975-8232.9(3).1167-75.

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