QUANTITATIVE PHYTOCHEMICAL PROFILE AND IN-VITRO ANTIOXIDANT POTENTIAL OF D5 CHOORANAM, AN ANTIDIABETIC SIDDHA FORMULATION
HTML Full TextQUANTITATIVE PHYTOCHEMICAL PROFILE AND IN-VITRO ANTIOXIDANT POTENTIAL OF D5 CHOORANAM, AN ANTIDIABETIC SIDDHA FORMULATION
Gayathri Gunalan *, R. Rathinamala, S. Sugin Lal Jabaris, S. Suganya, A. Durga, S. Selvarajan, K. Kanakavalli and A. Rajendra Kumar
Siddha Regional Research Institute (Central Council for Research in Siddha, Kuyavarpalayam, Puducherry, India.
ABSTRACT: D5 Chooranam is a classical Siddha formulation developed by Central Council for Research in Siddha, prescribed for the management of diabetes mellitus. The present work was carried out to assess phytochemical profile and also to explore the antioxidant potential of Siddha drug D5 Chooranam. The Phytochemical profile and antioxidant activity were evaluated in five different solvent extracts (Ethyl acetate, chloroform, ethanol, petroleum ether, and water) of D5 Chooranam. The phytochemical analysis of D5 Chooranam disclosed the existence of various phytochemicals in D5 Chooranam. The amount of phytochemicals present in D5 Chooranam also correlates with the various pharmacological activities as per its therapeutic indications. The considerable radical scavenging capacity of D5 Chooranam, particularly in its water extract, ethyl acetate extract, and fresh aqueous solution, is well justified by the results of all in-vitro antioxidant studies. These results reveal the beneficial effect of D5 Chooranam against oxidative stress associated disorders like diabetes mellitus and its complications. Hence it can be concluded that it may be due to the presence of vital phytochemicals in this exclusive novel blend of herbs and makes this formulation to be a virtuous choice for treatment of diabetes mellitus and its complications in Siddha clinical practice.
Keywords: Siddha medical system, D5 Chooranam, Free radicals, Phytochemicals, Diabetes mellitus
INTRODUCTION: Free radicals are the reactive oxygen species (ROS) produced during intracellular metabolic process or by exposure of human body to various exogenous agents like UV- radiation, pollutants, drugs and smoke 1. Body encounters the deleterious effects of such ROS by eliciting antioxidant defense system or by undergoing apoptosis.
Imbalance in body redox homeostasis results in “oxidative stress”, which is responsible for etiology of various diseases like Diabetes mellitus, atherosclerosis, cardiovascular diseases, inflammatory diseases, aging, etc.,2. DM is a metabolic disorder rising to an upsetting epidemic level. This is characterized by elevated level of blood glucose as a consequence of insulin deficiency or impaired insulin action or by both.
Prevalence of DM is increasing globally. It is predicted to rise 5.4% by 2025 of the world population 3. According to projections, 80% of the world's diabetic population will come from low- and middle-income nations by 2030. The dynamics of diabetes are changing quickly in these countries. Uncontrolled Diabetes mellitus leads to the development of various metabolic complications which involves macro vascular and micro vascular diseases. The most common complications are Diabetic retinopathy, Diabetic nephropathy, Diabetic neuropathy, cardiovascular diseases, and stroke 4, 5. The pathogenesis of these complications involves oxidative stress.
Although many oral hypoglycemic agents are available for the management of DM, due to limitations like high cost and undesirable adverse effects, more attention is being engrossed on identification of natural products with antidiabetic potential, strong antioxidant properties and low toxicity for discovery of ethnomedicines in the management of DM. Ethnomedicines are rich in phytochemicals like phenol, tannins, flavonoids, alkaloids, and a lot more responsible for various pharmacological activities. Siddha is a one of the earliest systems of medicine in India. The exclusivity of Siddha system is apparent by its constant service to manhood for more than 5000 years in tackling diseases and in maintaining its mental, physical, and moral health. Siddha system had been used by millions of people in the southern parts of India especially in Tamil Nadu and Kerala. Siddha system of medicine classifies diseases and disorders into 4448 types in which disease are being treated based on the three principles namely vadham, pittam, and kabam 1.
Siddha system of medicine deals with 11 metals, 64 pashanam, 120 uprasams and animal products in preparing medicines. Some Siddha formulations are composed of herbs with bioactive phytoconstituents, which possess wonderful antioxidant potential. D5 Chooranam is a one such classical polyherbal formulation composed of medicinal plants like Avaarai (Cassia auriculata), Kontrai (Cassia fistula), Naval (Syzygium cumini), Kadazhangil (Salacia oblonga), Korai kizhangu (Cyperus rotundus), Kostam (Costus speciosus), and Lavanga pattai (Cinnamomum zeylanicum) for the management of Type II Diabetes. Though all the ingredients were proved to have antidiabetic and various related pharmacological activity individually, the synergistic effect of all these medicinal plants in a single formulation may be evidenced in D5 Chooranam and hence it is an effective antidiabetic formulation used in the treatment of DM widely. Besides its wide usage for the treatment of DM, literatures for the elucidation of phytochemicals and antioxidant potential of D5 Chooranam are scanty. Hence, the current study was focussed to explore the phytochemical compositions and its antioxidant properties of D5 Chooranam.
METHODS AND MATERIALS:
Study Drug: The study drug, D5 Chooranam was purchased from Department of Pharmacy, Siddha Central Research Institute, Central Council for Research in Siddha, Arumbakkam, Chennai (Batch No: D505042021).
Chemicals: Fine chemicals like Quercetin, Curcumin, Nicotinamide Adenine Dinucleotide Hydrogen (NADH) used in this study were obtained from HiMedia Laboratories Private Limited (Mumbai, India). Chemicals such as N-(1-naphthyl) ethylenediamine dihydrochloride), Ascorbic acid, BHT (Butylated Hydroxy Toluene) and Phenazonium Methosulphate (PMS) were procured from LOBA chemie private limited (Mumbai, India). NBT (Nitro Blue Tetrazolium) and Gallic acid were of Sisco Research Laboratories Private Limited (Maharashtra, India) make. All other chemicals and reagents used in this study were of analytical grade.
Preparation of Extracts: Five different solvent extracts of Siddha drug, D5 Chooranam was prepared with increasing polarity namely Petroleum ether, Chloroform, Ethyl acetate, Ethanol and Water by cold percolation method 6. 100g of D5 Chooranam was soaked in 250ml of each solvent and kept aside in room temperature for 72 hours and mixed with glass rod on daily basis. Then the mixture was filtered using whatman No.1 filter paper. The filtrate was evaporated to dryness and stored at 4℃. The yield of drug extract was calculated and expressed by following the formula,
Yield of drug extract = Final amount of drug obtained / Initial amount of drug taken for extraction × 100
To prepare a 2 mg/ml of stock solution, 100mg of Chloroform, Petroleum Ether, Ethyl acetate, Ethanol and Water extract was weighed and dissolved individually in 50ml of Dimethyl Sulfoxide (DMSO) and stored in refrigerator for further analysis. In addition, an aqueous extract was also freshly prepared by dissolving 100mg of D5 Chooranam (drug) directly in 50ml of distilled water.
Phytochemical Analysis:
Qualitative Phytochemical Analysis: The standard procedures of Sofowara (1993), Trease and Evans (1989) and Harborne (1973) 7, 8, 12. were used for the analysis of D5 Chooranam extracts to confirm the presence of various phytochemicals like Alkaloids, Coumarins, Tannins, Saponins, Flavonoids, Quinones, Phenols, Carbohydrate, Proteins, Cardiac Glycosides, Gum, and Starch.
Quantitative Phytochemical Analysis:
Total Phenol Estimation: The phenol content in various D5 Chooranam extracts were determined by using the procedure of McDonald et al., (2001) 9. 200µl of different concentration of extracts were mixed with 1ml of Folin-Ciocalteau reagent (1:2 diluted) and 3ml of 20% sodium carbonate after incubation at room temperature in dark for 90 minutes, the absorbance of the reaction mixture was measured at 765nm using Microprocessor based Double beam UV-Visible Spectrophotometer (Igene Labserve, India). The resultant values were expressed in mg of Gallic acid equivalents (GAE)/g of drug.
Total Flavonoid Estimation: The flavonoid content was evaluated by Chang et al., (2002) 10. 0.25ml of different concentration of extracts were taken and the volume was made up to 1.25ml using distilled water. Into the mixture 75µl of sodium nitrate (5%) and 0.15ml of aluminum chloride (10%) solution was added and gently mixed. Finally, 0.5ml of 0.1M Sodium hydroxide was added, and the volume was made up to 2.5ml using distilled water. After the resulting solution shaken well, absorbance was measured at 510nm using Microprocessor based Double beam UV-Visible Spectrophotometer (Igene Labserve, India). Total flavonoid in D5 Chooranam was expressed in mg of Quercetin Equivalents (QE)/g of the drug.
Total Tannin Estimation: Peri and Pompei (1971) method as used for determination of tannin content in D5 Chooranam extracts. 1ml of various concentrations of drug extracts was taken and the volume was made up to 2 ml with distilled water. Then 0.5ml of Folin’s phenol reagent (1:2 diluted) and 5ml of 35% Sodium carbonate solution was mixed. The resulting mixture was incubated for 5mins at room temperature for the development of blue color. The color intensity was read at 640nm using Microprocessor based Double beam UV-Visible Spectrophotometer (Igene Labserve, India). The total tannin content was expressed as mg of Gallic acid Equivalents (GAE)/g of drug.
Alkaloid Estimation: The alkaloid content of D5 Chooranam was estimated by Harborne, (1973) 12. To 5g of D5 Chooranam, 200ml of 20% acetic acid was added and kept undisturbed for 4 hrs in a beaker covered with parafilm. Then the resulting mixture was filtered and heated in boiling water bath to concentrate it into one fourth of the volume. The alkaloids were precipitated by adding concentrated ammonium hydroxide solution in drop wise manner. After allowing the solution to settle down, precipitate was collected and weighed.
Antioxidant Assays: The antioxidant nature of D5 Chooranam was studied in terms of free radical scavenging activity. Various concentrations (10-1500µg) of D5 Chooranam extracts were analyzed for antioxidant activity in a dose specific manner.
Nitric Oxide Radical Scavenging Activity of D5 Chooranam: Nitric oxide radical scavenging potential of D5 Chooranam extracts were analyzed by Green et al., (1982) and Jagetia et al., (2004) 13 method. Nitric oxide produced in the reaction involving sodium nitroprusside and oxygen was measured by Griess reaction. Sulfanilamide (1%) and naphthyl ethylene diamine dihydrochloride (0.1%) in ortho-phosphoric acid (2.5%) were mixed thoroughly to prepare Griess reagent right before use.
The reaction mixture of 0.5ml of sodium nitroprusside in phosphate buffered saline and 1 ml of different concentration of D5 Chooranam extracts (20, 40, 60, 80,100µg) were incubated for 150 minutes at 25ºC. After incubation 0.5ml of reaction mixture was mix up with equal volume of fresh Griess reagent. The absorbance of the solution at 546nm was measured in Microprocessor based Double beam UV-Visible Spectrophotometer (Igene Labserve, India). Standard Ascorbic acid was also treated similarly. Percentage of inhibition was determined using the following formula:
Percentage (%) of nitric oxide radical scavenging assay = [(A0-A1)/A0] × 100
Superoxide Anion Radical Scavenging Activity of D5 Chooranam: Superoxide radical scavenging assay of D5 Chooranam extract were performed as mentioned by Nishi Miki et al., (1972) 14 method. Reaction solution was prepared by mixing 1ml of 156µM NBT solution (pH-7.4), 468µM NADH (pH-7.4) solution and 0.1ml of various concentrations of D5 Chooranam extracts ranging from 20-1500µg.
The reaction was then initiated by adding 100µl of PMS (pH-7.4) and incubated for 5 minutes at 25ºC. The absorbance of the sample mixture was read at 560nm against control using Microprocessor based Double beam UV-Visible Spectrophotometer (Igene Labserve, India). Quercetin was used as the standard, and it was also treated the same way. Percentage of inhibition was calculated according to formula as under:
Percentage (%) of superoxide scavenging = (A0 − A1)/A0 × 100
Hydroxyl Radical Scavenging Activity of D5 Chooranam: To assess the hydroxyl radical scavenging activity of five different solvent extracts of Siddha formulation D5 Chooranam, To 1ml of iron-EDTA solution, 0.5ml of EDTA (0.018%) and 1ml of DMSO were added to prepare the reaction mixture. The reaction was initiated by the addition of 0.5ml of 0.22% ascorbic acid solution. The experimental mixture was placed in water bath for 15 minutes at 80ºC-90ºC. To terminate the ongoing reaction 1ml ice-cold TCA was added to the final mixture containing 3ml of Nash reagent. After incubating at room temperature for 15 minutes, the developed yellow color was read spectrophotometrically at 412nm using Microprocessor based Double beam UV-Visible Spectrophotometer (Igene Labserve, India) 15. The activity of standard (BHT) was assayed similarly. The % of inhibition the test extract was calculated using the below cited formula:
Percentage (%) hydroxyl radical scavenging activity = [A0− (A1−A2] × 100/A0
Total Reducing Power Activity of D5 Chooranam: D5 Chooranam extracts were investigated for its total reducing power activity according to Oyaizu, (1986) 5 method with slight modification. The various concentrations (20, 40, 50, 80, 100, 150, 200, 250µg) of D5 Chooranam extracts were added with 2.5ml of phosphate buffer (pH-6.6) and 2.5ml of potassium ferricyanide (1%). After incubating at 50ºC for 20 minutes, 2.5ml of 10% TCA was added and the tubes were then centrifuged at 2500rpm for 10 minutes.
Followed by centrifugation, the resultant Supernatant was mixed with 2.5ml of distilled water and 0.5ml of 0.1% FeCl3. The absorbance of the experimental mixture was read at 700nm using Microprocessor based Double beam UV-Visible Spectrophotometer (Igene Labserve, India). BHT was treated as standard, and it was also treated similarly.
Statistical Analysis: All the values were expressed as Mean ± Standard Deviation (SD). All Statistical analysis including IC50 value determinations were performed using GraphPad Prism software (9.3.1). Results of the study was statistically analyzed using ANOVA (One-Way) followed by Least Significant Difference (LSD) test and the P value was calculated to ascertain the statistical significance of the observed changes. P<0.05 was considered as significant. All the assays were done in triplicates.
RESULTS:
Yield of D5 Chooranam Extracts: Five different solvent extracts of D5 Chooranam were prepared using solvents with increasing polarity like Water, Ethyl acetate, Chloroform, Petroleum ether, and Ethanol by cold percolation method. The yield of the extract was calculated and depicted graphically in Fig. 1.
The order of extract yield was found to be Water > Ethanol > Ethyl acetate > Chloroform > Petroleum Ether. It showed that the yield of water extract was comparatively higher than that of other extracts. This result infers the nature of phytochemicals present in D5 Chooranam as hydrophilic. As most of the physiological conditions are hydrophilic in nature, the bioavailability of the D5 Chooranam would be greater when consumed with its adjuvant, warm water and thereby it elicits its biological activity effectively.
FIG. 1: YIELD OF D5 CHOORANAM EXTRACTS IN PERCENTAGE
Qualitative Phytochemical Analysis: The results of preliminary phytochemical analysis were delineated in Table 1. The presence and absence of phytochemicals were illustrated as “+” (positive) and “-” (negative) respectively. Grades like 1+, 2+, 3+ were given to represent the degree of colour intensity compared with the other extracts. Qualitative phytochemical analysis of D5 Chooranam extracts of five different solvents has exhibited the presence of phytochemicals like Flavonoids, Quinones, Phenols, Alkaloids, Tannins, Coumarins, Saponins, and Cardiac Glycosides. The analysis of fresh aqueous solution also revealed the presence of Coumarins, Tannins, Saponins, Flavonoids, Quinones, Phenols and Cardiac Glycosides whereas, alkaloid, was found to be absent.
The existence of many phytochemicals in both water extract and fresh aqueous solution of D5 Chooranam correlates with the increased yield of water extract. Due to the presence of many phytochemicals like phenol, flavonoids etc, an increased quantity of water extract was resulted. Hence, it can be arrived that water extract is the best extract of D5 Chooranam drug. Presence of significant phytochemicals like Tannins, Flavonoids, Alkaloids, Phenols, Saponins and Cardiac glycoside were also confirmed in chloroform, ethyl acetate and ethanol extracts of D5 Chooranam. Whereas petroleum ether extract was found to contain tannins, phenols, flavonoids, quinones only.
TABLE 1: QUALITATIVE PHYTOCHEMICAL ANALYSIS OF D5 CHOORANAM
S. no. | Name of the
Test |
Petroleum
Ether Extract |
Chloroform
Extract |
Ethanol
Extract |
Ethyl Acetate Extract | Water
Extract |
Aqueous Drug Solution |
1 | Alkaloids | - | + | - | - | + | - |
2 | Coumarins | - | - | - | - | + | + |
3 | Tannins | + | + | + | + | + | + |
4 | Saponins | - | - | 2+ | - | + | + |
5 | Flavonoids | + | 2+ | + | + | 2+ | + |
6 | Quinones | 3+ | 3+ | - | - | 3+ | 2+ |
7 | Phenols | + | + | 3+ | 2+ | 3+ | 2+ |
8 | Carbohydrates | - | - | - | - | - | - |
9 | Proteins | - | - | - | - | - | - |
10 | Cardiac Glycosides | + | 2+ | 2+ | 2+ | 3+ | 2+ |
11 | Gum | - | - | - | - | - | - |
12 | Starch | - | - | - | - | - | - |
Quantitative Phytochemical Analysis: The amount of Phenols, Flavonoids, Tannins and Alkaloids in various solvent extracts were shown in Fig. 2A-C.
FIG. 2: QUANTITATIVE ESTIMATION OF TOTAL (A) TOTAL PHENOL (B) TOTAL FLAVONOID (C) TOTAL TANNIN CONTENT IN VARIOUS SOLVENT EXTRACTS OF D5 CHOORANAM
Total flavonoid, total phenol, and total tannin content of various solvents extracts of D5 Chooranam were calculated against the standard curve with R2 value of 0.997, 0.998, and 0.999 respectively. The estimation of phytochemicals in D5 Chooranam extracts revealed that total phenols, total flavonoids, and total tannin content was found to be present in high concentration in ethanol extract compared with the other extracts. Followed by ethanol extract, water extract was found to contain more phenol whereas it has lesser tannin and flavonoid content. Besides ethanol and water extract, appreciable amount of flavonoids and tannins were present in ethyl acetate extract of D5 Chooranam also. Whereas the chloroform extract of the study drug has exhibited decreased level of the phytoconstituents like phenol, flavonoid, and tannin, than water and ethyl acetate extract. Meanwhile, petroleum ether extracts notably contain least quantity of phenols, flavonoids, and tannins than above mentioned other four solvent extracts. Contrast to the estimation of other phytoconstituents, alkaloid content was quantified directly in D5 Chooranam without preparing any extract by gravimetric method of Harborne (1973). 0.323g of alkaloid was found to be present in 5 gram of drug and it was graphically presented in Fig. 3.
FIG. 3: QUANTITATIVE ANALYSIS OF ALKALOID CONTENT IN D5 CHOORANAM
Antioxidant Potential of D5 Chooranam: D5 Chooranam extracts were analyzed for the antioxidant potential using various in-vitro assays, like nitric oxide radical scavenging assay, Superoxide radical scavenging assay, Hydroxyl radical scavenging assay and total reducing assay. It was observed that various concentrations of drug extracts ranging from 10-1500µg/ml have scavenged the free radicals in dose specific manner in different methods. All the assays were done in triplicates.
Nitric Oxide Radical Scavenging Assay: Nitric oxide scavenging potential of D5 Chooranam extracts showed a variable level of inhibitory activity in a dose specific manner. For this assay, Ethanol extract, Ethyl acetate extract, Water extract and Aqueous extract were used in the concentration ranging from 20-100µg.
Whereas petroleum ether and chloroform extract were tested with 50-250µg concentration as they did not show any scavenging activity at lower concentration. The percentage of inhibition of various drug extracts were calculated against standard compound ascorbic acid using formula as mentioned under methods.
FIG. 4: NITRIC OXIDE RADICAL SCAVENGING POTENTIAL OF (A) ETHANOL AND ETHYL ACETATE EXTRACTS OF D5 CHOORANAM. (B) WATER EXTRACT AND AQUEOUS SOLUTION OF D5 CHOORANAM. (C) PETROLEUM ETHER AND CHLOROFORM EXTRACT OF D5 CHOORANAM. (D) IC50 VALUE OF SEVERAL D5 CHOORANAM EXTRACTS. (ASCORBIC ACID WAS USED AS STANDARD)
The IC50 value of water extract was found be 15.5µg/ml, which was lesser than that of ascorbic acid (standard) IC50 value (24.29µg/ml). From this result, it was evident that D5 Chooranam acts as a strong nitric oxide radical scavenger even than the standard ascorbic acid. The result was also statistically significant (P<0.001) and it was presented in Fig. 4A, 4B, 4C & 4D.
The IC50 value of other extracts like ethyl acetate, ethanol, and aqueous solution was found to be 37.53 µg/ml, 43.61 µg/ml, and 55.68µg/ml respectively. Higher IC50 values were observed for chloroform extract and petroleum ether extract (158.86 µg/ml and 190.81 µg/ml) respectively and hence they have least nitric oxide radical scavenging activity.
All the results were statistically significant with a P value less than (0.001).
Superoxide Scavenging Assay: Superoxide scavenging potential of D5 Chooranam extracts were studied against standard curcumin. Percentage of radical scavenging activity and IC50 values of D5 Chooranam extracts against superoxide radicals were depicted in Fig. 5A-E. Ethyl acetate extract showed the excellent superoxide scavenging activity with a low IC50 value of 49.41µg/ml. It clearly depicted that its activity is almost near to the standard Curcumin (IC50 = 15.26 µg/ml). Followed by Ethanol extract, Aqueous extract, water extract and Chloroform extract was found to possess appreciable superoxide scavenging activity with IC50 value of 72.63 µg/ml, 87.30 µg/ml, 96.52 µg/ml, and 150.91 µg/ml respectively.
FIG. 5: SUPEROXIDE RADICAL SCAVENGING ASSAY OF (A) AQUEOUS SOLUTION AND WATER EXTRACT. (B) ETHANOL AND ETHYL ACETATE EXTRACT (C) CHLOROFORM EXTRACT (D) PETROLEUM ETHER EXTRACT (E) IC50 VALUE OF VARIOUS D5 CHOORANAM EXTRACTS. (CURCUMIN WAS USED AS STANDARD)
Compared to the other extracts of D5 Chooranam, Petroleum ether extract exhibited the minimal level of superoxide radical scavenging activity with very high IC50 value. (561.91µg/ml). Results of all these assays were statistically significant with P value less than 0.001.
Hydroxyl Radical Scavenging Assay: The hydroxyl radical scavenging potential and IC50 of various solvent extracts of D5 Chooranam was presented in the Fig. 6A-D. Despite the results of other assays, Petroleum ether extract, and Ethyl acetate extract exhibited high hydroxy radical scavenging activity with an IC50 value of about 25 µg/ml for each which is less than the IC50 value of BHT (125 µg/ml). Results of this study demonstrated the strong hydroxyl radical scavenging potential of D5 Chooranam.
In addition, ethanol extract and water extract showed the satisfying level of radical scavenging activity with an IC50 value of 62.5µg/ml and 70µg/ml respectively. Whereas, chloroform extract exhibited low scavenging activity with very high IC50 value (500 µg/ml).
FIG. 6: HYDROXYL RADICAL SCAVENGING ASSAY OF (A) PETROLEUM ETHER AND ETHYL ACETATE EXTRACT (B) ETHANOL AND CHLOROFORM EXTRACT (C) AQUEOUS AND WATER EXTRACT (D) IC50 VALUE OF VARIOUS D5 CHOORANAM EXTRACTS (BHT AS STANDARD)
Total Reducing Power Assay: D5 Chooranam extracts subjected to the total reducing power assay demonstrated various levels of reducing power activity. The results were statistically significant (P<0.001) presented in Fig. 7A-C. BHT was used as a standard drug.
FIG. 7: TOTAL REDUCING POWER ASSAY OF (A) ETHANOL, ETHYL ACETATE AND WATER EXTRACT. (B) PETROLEUM ETHER EXTRACT, CHLOROFORM EXTRACT AND AQUEOUS SOLUTION (C) IC50 VALUE OF VARIOUS D5 CHOORANAM EXTRACT (BHT AS STANDARD)
When compared to the other solvent extracts of D5 Chooranam, ethanol extracts showed the maximum reducing power activity with IC50 values of 17.05 µg/ml, and this was followed by Ethyl acetate extract, Aqueous extract, Water extract and Petroleum ether extract with an IC50 value of 24.05 µg/ml, 26.93 µg/ml, 40 µg/ml and 106.27 µg/ml respectively. Contrast to all other in-vitro assays, D5 Chooranam extracts exhibited good reducing power activity than that of standard drug, BHT (IC50=125.06 µg/ml).
DISCUSSION: In the modern era, poly herbal preparations have gained greater importance than ever before, mainly due to their efficacy, less side effects and easy availability. As claimed by various research outcome, these polyherbal formulations are more advantageous than single herbal concentrations since they have enhanced synergistic actions of various ingredients. Plant produced. Plant produced secondary metabolites, are the main source of natural antioxidant that can eliminate the harmful reactive oxygen species. Antioxidants quench the action of free radicals, which are responsible for the etiology of various diseases particularly DM, neurodegenerative disorder, inflammation, and cancer. Free radicals are produced during the metabolic process leading to several clinical implications. Even though many synthetic antioxidants are available to deal the oxidative stress, due to the adverse effects research interest has been developing on the natural antioxidants. In the present study, D5 Chooranam a classical Siddha formulation was screened for phytoconstituents and also for its antioxidant potential to justify its therapeutic potential as an antidiabetic drug. In the drug extraction process, the yield of water extract was higher (7.67%) when compared to the other extracts of D5 Chooranam. In Siddha clinical practice D5 Chooranam was prescribed along with warm water as an adjuvant for diabetic patients. High yield of water extract of D5 Chooranam clearly depicted that the bioavailability of D5 chooranam at its therapeutic dose would be more under physiological conditions and thus it would be of much beneficial in combating Diabetes mellitus. Hence, the adjuvant (warm water) is a perfect choice for the absolute therapeutic benefit of D5 chooranam.
It is vital to monitor the phytoconstituents of D5 Chooranam to evaluate their efficacy and safety for therapeutic use. Phytochemical assessment of various solvent extracts of D5 Chooranam disclosed the existence of phenols, flavonoids, alkaloids, coumarins, tannins, saponins, quinones, carbohydrates, proteins, and cardiac glycosides. No previous studies about the presence of these phytoconstituents in the different solvent extracts of D5 Chooranam have been observed in the literature and hence this study is first of this kind. Phenol and flavonoid compounds are the plant secondary metabolites. They hold an aromatic ring with hydroxyl group and have been reported to possess anti-diabetic, cardio protective, antioxidants, anticancer, antibacterial, anti-inflammation, immune promoting and skin protection properties 16, 17. In the quantitative analysis, presence of considerable number of bioactive compounds like phenols, flavonoids, tannins and alkaloids in ethanol extract exhibits the antioxidant potential of D5 Chooranam and their benefits on health. Reactive Oxygen Species (ROS) namely superoxides, peroxides, hydroxyl radicals and singlet oxygen are the derivatives of oxygen which are continuously produced under physiological condition due to exposure of exogenous chemical and/or several endogenous chemical reactions that involves redox enzymes and bioenergetics electron transfer 18. As a result of overproduction of ROS and/or insufficient antioxidant defence mechanism, there is an increase of ROS and which in turn leads to oxidative stress. Compared to the synthetic antioxidants, plants possess good antioxidant ability and are safer too. Various mechanisms like Inhibition of chain initiation, decomposition of peroxides, transition metal ion catalysts binding, reductive strength, and radical scavenging potential attributes to antioxidant activity. Extensive study on free radical scavenging potential provides the logical basis behind the unexplored properties like antioxidant activity of any natural product/drug.
In the present study, four different antioxidant methods for assessment of antioxidant activity have been studied. Various concentrations ranging from 20 - 100 µg/mL of D5 Chooranam were assayed for their antioxidant activity using different in vitro models. Extracts of D5 Chooranam presented notable level of scavenging activities against the superoxide radicals, nitric oxide radicals and hydroxyl radicals in a dose specific manner in the various methods.
Nitric oxide (NO) is a radical produced by phagocytic cells and endothelial cells; it is involved in the management of several physiological processes 19. Several diseases are associated with increased concentration of NO 20 and it plays substantial role in inflammatory process. Peroxynitrite anions are the free radicals produced during the reaction between oxygen and excessive nitrite, nitric oxide 21. Nitric oxide can rapidly react in the physiological conditions to form nitrates, nitrites and s-nitrosothiols. These compounds play a crucial role in intermediating various xenotoxic effects such as DNA damage through peroxynitrite. In the present study, nitric oxide radical assay of D5 Chooranam was performed in all the 5 different solvent extracts. On comparative analysis with other extract, water extract of D5 Chooranam showed maximum nitric oxide radicals scavenging activity with IC50 value of 15.5µg/ml against the standard ascorbic acid whose IC50 value was found to be 24.29µg/ml. This outcome demonstrated that D5 Chooranam's antioxidant properties when combined with its adjuvant water. Oxidative enzymatic reaction such as autoxidation by catecholamine generates Superoxide anion from molecular oxygen 22. Among free radicals Superoxide is a primary oxygen radical that is produced when an oxygen molecule receives one electron. The scavenging activity in relation to the superoxide radical is estimated in terms of suppression of generation of O2-. Superoxide anion acts as predecessors of hydroxyl radicals and singlet oxygen by initiating lipid peroxidation indirectly via superoxide and hydrogen peroxide 23. The antioxidant nature of flavonoids is effectively elucidated via the scavenging of superoxide anion 24. As D5 Chooranam is rich in flavonoids, it exhibits an appreciable scavenging action towards superoxide radicals.
Ethyl acetate extract exhibited maximum superoxide scavenging activity with half maximal inhibitory concentration value of 65.12 µg/ml compared to that of the standard curcumin with half maximal inhibitory concentration value of 15.26 µg/ml. The outcome of the study has showed the potential antioxidant behaviour of D5 Chooranam. Being a polar solvent, much water-soluble phytonutrients were present in the ethyl acetate extract of D5 Chooranam and hence the antioxidant capacity of therapeutic dose is justified.
In biological systems, hydroxyl radicals are formed as an extremely reactive species and have been responsible for free radical pathogenesis by damaging each and every molecule within any living cells 25. This radical triggers DNA strand breakage by its capacity to join nucleotides and thus results in cytotoxicity, mutagenesis, and carcinogenesis. Additionally, this species is predicted as one of the lipid peroxidation process initiators and it also abstract H+ ions from unsaturated fatty acids. In the current study, hydroxyl radical scavenging activity of D5 Chooranam extracts were compared with standard BHT. Among the other extracts, ethyl acetate and petroleum extract were found to be superior with same half maximal inhibitory concentration value of 25µg/ml, whereas standard BHT has IC50 of 125µg/ml. This indicates the excellent antioxidant activity of D5 Chooranam. Ethanol extract of D5 Chooranam exhibited excellent reducing power activity with IC50 value of 17.05µg/ml compared with standard BHT with IC50 value of 125.06µg/ml. which in turn represents potential metal chelating activity of D5 Chooranam. The presence of large amounts of phenols and flavonoids in D5 Chooranam was abundantly supportive of the observed antioxidant property. The strong antioxidant activity demonstrated by D5 Chooranam would possibly bring about the reduction of oxidative stress in diabetes patients.
CONCLUSION: Thus, our study findings demonstrate the antioxidant potential of the various D5 Chooranam extracts. Qualitative and quantitative assessment of various D5 Chooranam extracts showed the presence of significant number of various phytochemicals. In-vitro antioxidant analysis of D5 Chooranam also shows an antioxidative activity in most of the oxidative stress-related parameters. Though the individual ingredients of the study drug have been proved individually for diverse biological activity, the synergetic effect of those phytoconstituents in this formulation could be reason for its significant free radical scavenging activity. Hence, it can be concluded that D5 Chooranam has potent antioxidant activity against various free radicals which were generated during diverse metabolic reaction. Further detailed investigations are required to completely elucidate the cellular mechanism of D5 Chooranam on various disease conditions like DM which results from oxidative stress.
Source of Funding: This research project was funded by Ministry of Ayush, Government of India, under the AYURGYAN Scheme’s component, Research & Innovation in Ayush (erstwhile Extra Mural Project scheme) via Sanction No: S-14014/11/2019-scheme dated 25.02.2020.
ACKNOWLEDGEMENTS: Authors express their sincere gratitude to EMR Section, Ministry of Ayush, Government of India, for the support and financial assistance during the course of this research work. Authors extend their sincere thanks to Technical Section of CCRS for their constant support and cooperation. Authors are also thankful to The Assistant Director & I/c and Department of Pharmacy, Siddha Central Research Institute for their efforts in the preparation of trial drug, D5 Chooranam.
CONFLICTS OF INTEREST: The authors declare that they have no Conflicts of interest.
REFERENCES:
- Kiruthika B, Abinaya R, Karolin Daisy Rani R and Saravana Devi MD: A Study on phytochemical properties and Antioxidant activity of different solvent extract of Siddha drug Paruthi Chooranam in In-vitro model. International Journal of Advanced Research in Biological Sciences 2019; 6(2): 51-61.
- Sougata Ghosh, Abhishek Derle, Mehul Ahire, Piyush More, Soham Jagtap, Suvarna D. Phadatare, Ajay B. Patil, Amit M. Jabgunde, Geeta K. Sharma, Vaishali S. Shinde, Karishma Pardesi, Dilip D. Dhavale and Balu A. Chopade: Phytochemical Analysis and Free Radical Scavenging Activity of Medicinal Plants Gnidia glauca and Dioscorea bulbifera. PLoS ONE 2013; 8(12): 82529.
- Taofik Olatunde Sunmonu and Francis Bayo Lewu: Phytochemical analysis, in-vitro antioxidant activity and inhibition of key diabetic enzymes by selected nigerian medicinal plants with antidiabetic potential. Indian Journal of Pharmaceutical Education and Research 2019; 53(2): 250-260.
- Edwin N Frankel and Bruce German J: Antioxidants in foods and health: problems and fallacies in the field. Journal of the Science of Food and Agriculture 2006; 86(13): 1999–2001.
- Razieh Yazdanparast, Seifollah Bahramikia and Amin Ardestani: Nasturtium officinale reduces oxidative stress and enhances antioxidant capacity in hypercholesterolaemic rats. Chemico-Biological Interactions 2008; 172(3): 176–184.
- Veeramuthu Duraipandiyan, Muniappan Ayyanar and Savarimuthu Ignacimuthu: Antimicrobial activity of some ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India. BMC Complementary Medicine and Therapies 2006; 6: 35.
- Sofowora A: Medicinal plants and Traditional Medicine in Africa. Ibadan, Spectrum Books Ltd Pub 1993; 191-289.
- Trease GE and Evans WC: Pharmacognosy. Bailliere Tindall, London, Edition 1989; 11: 45-50.
- Suzy McDonald, Paul D Prenzler, Michael Antolovich and Kevin Robards: Phenolic content and antioxidant activity of olive extracts. Food Chemistry 2001; 73(1): 73-84.
- Chang CC, Yang MH, Wen HM and Chern JC: Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis 2002; 10(3): 178-182.
- Peri C and Pompei C: Estimation of different phenolic groups in vegetable extracts. Phytochemistry 1971; 10(9): 2187-2189.
- Harborne JB: Phytochemical Methods. London, Chapman and Hall Ltd, Edition 1973; 3: 49-188.
- Ganesh Chandra Jagetia, Shaival Kamalaksh Rao, Manjeshwar Shrinath Baliga and Kiran S Babu: The evaluation of nitric oxide scavenging activity of certain herbal formulations in-vitro: a preliminary study. Phytotherapy Research 2004; 18(7): 561-565.
- Morimitsu Nishikimi, Appaji Rao N and Kunio Yagi: The occurrence of superoxide anion in the reaction of reduced phenazine methosulphate and molecular oxygen. Biochemical and Biophysical Research Communications 1972; 46(2): 849-854.
- Kalaisezhiyen Pavithra and Sasikumar Vadivukkarasi: Evaluation of free radical scavenging activity of various extracts of leaves from Kedrostis foetidissima (Jacq) Cogn. Food Science and Human Wellness 2015; 4(1): 42-46.
- Makoto Oyaizu: Studies on Products of Browning Reactions: Antioxidative Activities of Product of Browning Reaction Prepared from Glucosamine. Japanese Journal of Nutrition and Dietetics 1986; 44(6): 307-315.
- Edwin N Frankel and Bruce German J: Antioxidants in foods and health: problems and fallacies in the field. Journal of the Science of Food and Agriculture 2006; 86(13): 1999–2001.
- Jiang ZY, Hunt JV and Wolff SP: Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxides in low density lipoproteins. Analytical Biochemistry 1992; 202(2): 384-389.
- Silva CG, Herdeiro RS, Mathias CJ, Panek AD, Silveira CS, Rodrigues VP, Renno MN, Falcao DQ, Cerqueira DM, Minto ABM, Nogueira FLP, Quaresma CH, Silva JFM, Menezes FS and Eleutherio ECA: Evaluation of antioxidant activity of Brazilian plants. Pharmacological Research 2005; 52(3): 229–233.
- Kumar RS, Sivakumar T, Sunderam RS, Gupta M, Mazumdar UK, Gomathi P, Rajeshwar Y, Saravanan S, Kumar MS, Murugesh K and Kumar KA: Antioxidant and antimicrobial activities of Bauhinia racemosa L. stem bark. Brazilian Journal of Medical and Biological Research 2005; 38(7): 1015-1024.
- Ialenti A, Moncada S and Di Rosa M: Modulation of adjuvant arthritis by endogenous nitric oxide. British Journal of Pharmacology 1993; 110(2): 701-706.
- Hemmani T and Parihar MS: Reactive oxygen species and oxidative DNA damage. Indian Journal of Physiology and Pharmacology 1998; 42(4): 440-452.
- Okuda T, Kimura Y, Yoshida T, Hatano T, Okuda H and Arichi S: Studies on the activities of tannins and related compounds from medicinal plants and drugs. I. Inhibitory effects on lipid peroxidation on mitochondria and microsomes of liver. Chemical and Pharmaceutical Bulletin 1983; 31(5): 1625-1631.
- Robak J and Gryglewski RJ: Flavonoids are scavengers of superoxide anions. Biochem Pharma 1988; 37(5): 837-41.
- Kazuko Shimada, Kuniko Fujikawa, Keiko Yahara and Takashi Nakamura: Antioxidative properties of xanthan on autooxidation of soybean oil in cyclodextrin. Journal of Agricultural and Food Chemistry 1992; 40(6): 945 - 948.
How to cite this article:
Gunalan G, Rathinamala R, Jabaris SSL, Suganya S, Durga A, Selvarajan S, Kanakavalli K and Kumar AR: Quantitative phytochemical profile and in-vitro antioxidant potential of D5 chooranam, an antidiabetic Siddha formulation. Int J Pharm Sci & Res 2023; 14(9): 4461-73. doi: 10.13040/IJPSR.0975-8232.14(9).4461-73.
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IJPSR
Gayathri Gunalan *, R. Rathinamala, S. Sugin Lal Jabaris, S. Suganya, A. Durga, S. Selvarajan, K. Kanakavalli and A. Rajendra Kumar
Siddha Regional Research Institute (Central Council for Research in Siddha, Kuyavarpalayam, Puducherry, India.
ggsrri16@gmail.com
06 January 2023
25 April 2023
13 July 2023
10.13040/IJPSR.0975-8232.14(9).4461-73
01 September 2023