Evaluation of Pharmacological Properties of Caesalpinia bonducella Seed and Shell Extract

Background: Caesalpinia bonducella L. is a medicinal plant belonging to the family Caesalpiniaceae. It is a prickly shrub widely distributed all over the world especially in Indian tropical regions such as Kerala, Andaman and Nicobar Islands and Sri Lanka. There are claims that its leaves or seeds/ seed kernel possess antipyretic, antidiuretic, antibacterial, antiviral, antiestrogenic and antidiabetic activities. Due to the above properties several preparations of the plant were used in folk medicine. Materials and Methods: The aqueous extract of Caesalpinia bonducella nut containing the seed and the shell, has been evaluated for qualitative analysis of secondary metabolites (tannis, flavonoids, alkaloids, saponins, coumarins, quinone and phenols), in-vitro anti-inflammatory, anti-diabetic assay, antioxidant, antimitotic and antimicrobial activity. The studies were carried out using HRBC membrane stabilization, inhibition of alpha amalyse enzyme, DPPH method, green gram growth inhibition, agar diffusion method respectively. Results: Our results indicate the presence of Alkaloids, Flavanoids and Saponins. We report in our study the antidiabetic, anti-inflammatory, anti-oxidant, anti-microbial and antimitotic activity of Caesalpinia bonducella.


INTRODUCTION
2][3] C. bonducella has been known to be used by Siddha practitioners in Malabar regions for psoriasis treatment. 1. bonducella is a large prickly shrub known to be a native of South India, Burma and Ceylon, particularly along the sea coast and up to 2500 ft. in hilly regions.4 It is reported in literature that most parts of the plant has therapeutic properties, but much has been studied with the seed and shell.[5][6] The alkaloids in Caesalpinia bonducella L are known to be found in shell, seed and twigs, the predominant one being Natin.The active molecule, Bonducin is reported to be present in the seed as a powerful glycoside.Saponins and terpenoids are also known to be found in seed.7 The shell is known to contain fatty oil, starch, sucrose, phytosterols, stearic, palmitic, oleic, linoceric, linolenic and a mixture of unsaturated acid of low molecular weights.The protein and amino acid content varies from 7.430 to 25.346%.8 The seeds are reported to have anti-diabetic properties.Type 2 diabetes, a chronic metabolic disorder affects people of all ages across the globe.This disease is characterized by increase in the blood glucose level which may be multifactorial. T primary cause is the decrease or lack of insulin production.The treatment regimen for Type 2 Diabetes is mainly to prevent breakdown of carbohydrates to glucose and preventing its diffusion into the intestinal membrane into blood stream.The abundance of natural resources in India and the rising numbers of Diabetes patients will pave the way for newer medications/adjunct therapies to manage the disorder.9 Inflammation is often associated with pain and involves the increase of vascular permeability, increase of protein denaturation and membrane alteration.When the cell undergoes injury, inflammation of tissue becomes a defensive response characterized by redness, pain, heat and swelling and loss of function in the injured area.The management of inflammation related diseases is of concern and may have to be addressed using plant extracts.10 The plant is also known to possess antioxidant, 11 antifilarial activity, 12 anticonvulsive activity 13 and anti-microbial activity, 14 antimalarial activity 15 antitumor activity 16 anti-ulcer activity 17 immunomodulatory activity 18 and anticataract activity. 19 ith the rising problems of Diabetes, inflammation related diseases and cancers; there is a need to address the issues using alternate therapy.As there is limited study on the aqueous extract of Caesalpinia bonducella L, shell and seed we have evaluated their pharmacological properties.

Plant Material
Nuts of the C. bonducella were collected from the local market in Bengaluru, Karnataka, India.The seed and shell extract was prepared by following the method of Shukla et al. 20 The nuts were shade dried, coarsely powdered and sieved to get a uniform powder.The sample was extracted in water using Soxhlet extractor.The crude extract obtained was evaporated and concentrated.

Phytochemical analysis
Phytochemical analysis was carried out for saponins, flavonoids, quinones, alkaloids and tannins were performed as described by Maria Shabbir et al. 21agner's reagents was used for alkaloid, foam test for saponins, lead acetate test for flavonoids, Braemer's test for tannins, Sulphuric acid test for quinones.All these experiments were carried out for water extract for seed and shell individually.

In-vitro Anti Inflammatory Assay
The activity was carried out by the method of Gandhisan et al. 22 The blood was collected from healthy volunteers and mixed with equal proportion of Alsever solution (2% dextrose, 0.8% sodium citrate, 0.5% citric acid and 0.42% NaCl).The sample was centrifuged at 3,000 rpm and cells washed with saline.
Extract concentrations of 200, 400, 600, 800 and 1000μg/ml was prepared using distilled water.To this 1 ml of extract, 1 ml of phosphate buffer, 2 ml hypo saline and 0.5ml of HRBC suspension were added.It was incubated at 37°C for 30 min and centrifuged at 3,000 rpm for 20 min.The percentage of HRBC membrane stabilization or protection was calculated by using the following Formula with Aspirin (1 mg/ml) as reference standard drug % protection = 100-(Optical density of drug treated sample/ Optical density of control) x100

Anti-Diabetic Assay
The anti-diabetic assay was carried out using 100 μl of (500,1000μg/ml) plant extracts and 200 μl of amylase and incubated at 37°C for 20 min.To the reaction mixture 1% starch (100 μl) was added and incubated at 37°C for 10 min.The reaction was arrested by adding 200 μl DNSA and keeping in a boiling water bath for 5 min.The reaction mixture was diluted with 2.2 ml of water and absorbance read at 540 nm against blank. 23

Determination of DPPH free radical scavenging activity
The antioxidant property was assessed using Ascorbic acid as the standard and DPPH (1,1-diphenyl-2-picrylhydrazyl) as control.100μl of the extract was taken with 3ml of DPPH solution and incubated for 30 min.The absorbance was read at 517nm and the ability of the DPPH to scavenge free radical was calculated using the formula DPPH scavenged (%) = {(Ac -At)/Ac} x 100 Where Ac is the absorbance of the control reaction and At is the absorbance in presence of the sample.The antioxidant activity of the extract was expressed as IC 50 which is the concentration in mg of dry material per ml (mg / ml) that inhibits the formation of DPPH radicals by 50%.Each value was determined from regression equation. 24

Anti-mitotic activity
Green gram seeds of equal weight were germinated in 500 μl of (10, 20, 30, 40 mg/ml) plant extracts in a 24-well microtiter plate.Seeds germinated in distilled water served as the control and that in drug doxorubicin as the standard (Kumar and Singhal, 2009).For the morphological study, the length of the radical was observed.Experiment was performed in triplicates. 25

Anti-Microbial Activity
The antimicrobial activity of the extract was assessed using Staphylococcus aureus, Candida albicans and Mycobacterium smegmatis by the agar diffusion method.The agar diffusion method was used to evaluate the anti-microbial activity of the plant extract.30 ml of nutrient agar was poured into petri plates containing 100µl of microorganisms (McFarlands Number 5).After 24 h the zone of inhibition was measured and compared using Streptomycin and Candid B as the standards for bacteria and fungi respectively.

Phytochemical analysis
The qualitative analysis of the secondary metabolites like tannins, flavonoids, alkaloids etc., was done for water extract of C. bonducella and the results tabulated in Table 1.Our results indicate the presence of flavanoids and alkaloids in both parts of the nut.The seed and shell was rich in saponins indicating its therapeutic value.

In-vitro Anti Inflammatory Activity
The HRBC membrane stabilization method was used to study the antiinflammatory activity.The prevention of hypo tonicity induced HRBC membrane lysis was taken as measure in estimating the anti-inflammatory property.The % protection is indicated in Figure 1 and depicted in Table 2.The maximum anti-inflammatory activity of seed and shell was found to be 84.37% and 91.304% respectively at 1000 µg/ml and the % protection of shell was close to that of standard drug aspirin.

Anti-Diabetic Assay
Inhibition of α-amylase is considered a strategy for the treatment of disorders in carbohydrate uptake such as diabetes and obesity.α-amylase activity can be measured in-vitro by hydrolysis of starch in presence of α-amylase enzyme.Glycomet GP2 was used as the standard drug.The results of the Anti-diabetic activity is tabulated in Table 3.The maximum activity was at a concentration of 1000µg/ml and shell showed a better antidiabetic activity compared to the seed.It is also observed that the antidiabetic activity of the shell extract is close to the standard value.

Determination of Antioxidant Efficacy by DPPH Method
1,1-Diphenyl-2-picrylhydrazyl is a stable free radical with red colour (absorbed at 550nm).If free radicals have been scavenged, DPPH will change its colour to yellow.This assay uses this character to show free radical scavenging activity.The seed did not show any anti-oxidant activity.% antiradical activity of the sample is indicated in Table 4.The IC 50 value was calculated and represented in Figure 2.

Anti-Mitotic Assay
Seeds of equal weight were taken in each well and 500µl of the extract of various concentrations were added.The dry weight of the seeds was taken after 24h and 48h.Doxorubicin (1mg/ml) was used as the standard drug and it showed 20% inhibition after 24h and 51.1% inhibition after 48hrs.Shell did not show any anti-mitotic activity.The results are tabulated in Table 5. Maximum inhibition of growth was found at 40mg/ml of seed extract as seen in Figure 3 and 4.   results indicate the use of C bonducella as an adjunct therapy for inflammation and diabetes.

Anti-Microbial Studies
The antimicrobial activity was assessed for the aqueous extract of shell using the agar diffusion method.The zone of inhibition was measured using streptomycin and Candid B as the standard for bacteria and fungi respectively.The results are indicated in Table 6 and Figure 5.Our results indicate that the crude extract of the sample showed a better antimicrobial activity, indicating its use as a topical application.

DISCUSSION AND CONCLUSION
Many herbal remedies have been employed in various medical systems for the treatment and management of different diseases.The plant Caesalpinia bonducella (syn: Caesalpinia crista Linn.) has been used in different system of traditional medication for the treatment of diseases and ailments of human beings. 26Phytochemicals are a class of molecules found predominantly in tea, grapes, berries, cocoa and other plants.
These are known to have diverse pharmacological properties. 27Though they do not have any nutritive value the protective and disease preventing properties have been well explored.It is in this context that the study of the pharmacological properties of Caesalpinia bonducella was conceived. 28Flavonoids are found in fruits, nuts, grains and vegetables and used extensively to study their effect on heart diseases and cancer.Flavanoids are known to exhibit anti-inflammatory and anti-oxidant and anti-microbial properties.This is in accordance with our results wherein the flavonoid content in the seed and shell extract are moderately high.Taken together, these results indicate the anti-oxidant and anti-inflammatory properties of Bondoc nut.Our results reveal the antimitotic activity of the seed extract and hence may be exploited further for the treatment of cancer.Our experimental results also reveal the inhibition of α amylase activity, indicating that the plant may be used in anti-diabetic therapy.Further, our results indicate the presence of Alkaloids, Flavanoids and Saponins.We report in our study the antidiabetic, anti-inflammatory, anti-oxidant, anti-microbial and anti-mitotic activity.Taken together our

Figure 2 :Figure 3 :Figure 4 :
Figure 2: IC 50 value of the aqueous extract of shell.The IC 50 was found to be 350.638µg/ml for the shell extract.(source: Personal collection)

Figure 5 :
Figure 5: A: Zone of inhibition for Candida albicans B: Zone of inhibition for Staphylococcus aureus C: Zone of inhibition for Mycobacterium smegmatis.(source: Personal collection)