Antioxidant and Possible Anti-Inflammatory Potential of Selected Medicinal Plants Prescribed in the Indian Traditional System of Medicine

Abstract

The 50% alcohol extracts of Swertia chirayita. (Roxb. ex Flem) (Gentianaceae), Cedrus deodar. (Roxb. ex Lamb) (Pinaceae), Boerhavia diffusa. (Linn.) (Nyctaginaceae), Berberis aristata. (Linn.) (Berberidaceae), Withania somnifera. Dunal (Solanaceae), Pongamia glabra. (Vent) (Papilionoideae), Petrocarpus santalinus. (Linn.) (Papilionoideae), Tinospora cordifolia. (Willd) Miers (Menispermaceae), and Acrous calamus. (Linn.) (Araceae), were screened for antioxidant and possible anti-inflammatory potential. The evaluation of antioxidant potential was determined by performing 2,2-diphenyl-1-picryl hydrazine (DPPH^˙) reduction assay, OH radical (OH^˙) scavenging activity, estimation of vitamin C, total polyphenols, and inhibition of polyphenol oxidase (PPO) as a model oxidizing enzyme. To understand the possible anti-inflammatory potential of the selected plants, trypsin and β.-glucuronidase inhibition assays were carried out. All the plants under study were found to be effective scavengers of DPPH radicals. Among the tested plants, only Pongamia glabra, Petrocarpus santalinus, Tinospora cordifolia., and Acrous calamus. were found to possess OH radical scavenging activity. Appreciable amounts of vitamin C and phenolics were estimated from all the plant extracts under study. Except for Pongamia glabra., all the tested plants demonstrated significant PPO inhibitory activity. The plants under study were found to have moderate antiproteolytic activity toward trypsin-induced hydrolysis of bovine serum albumin. Except for Cedrus deodar. and Acrous calamus., none of the plant extracts showed β.-glucuronidase inhibitory potential. The results of the current study may be of use for standardization and validation of herbal drugs containing the above mentioned plants as one of the ingredients.

Keywords:

• Anti-inflammatory activity
• antioxidants
• Ayurveda
• enzyme inhibition
• traditional medicinal plants

Introduction

Traditional Indian medicine, Ayurveda, has a long history and is one of the great living traditions. Considerable research on pharmacognosy, chemistry, pharmacology, and clinical therapeutics has been carried out on Ayurvedic medicinal plants of India (Patwardhan, 2005). An enormous body of research supports the recommendations that antioxidant therapy is an attractive alternative approach for amelioration of diverse ailments such as cancer (Papageorgious et al., 2005), cardiovascular diseases (Nuttal et al., 1999), inflammation, shock ischemia injury (Salvatore et al., 2001), diabetes, cataract (Kaneto et al., 1999; Kowluru et al., 2001), Alzheimer disease (Jose et al., 2004), and acute central nervous system injury (Yossi et al., 2002). Hitherto, antioxidants have been considered as one explanation for the beneficial effects of the healthy, Mediterranean-type diet (Nuttall et al., 1999).

Traditional medicine is an evolutionary process as communities and individuals continue to discover new techniques that can transform practices. Ethnopharmacology and drug discovery using natural resources remain important issues in the current target-rich, lead-poor scenario (Patwardhan et al., 2004). The current study is an attempt to strengthen the standardization of selected medicinal plants having common prescription in the Indian traditional system of medicine.

A brief overview of the traditional therapeutic applications of the selected plants under study is described here so as to understand their medicinal importance.

Swertia chirayita.: Whole plant, either powder, infusion, or as an extract, is used as an antidiarrheal, antimalarial, anthelmintic, and as a special remedy for bronchial asthma and liver disorders. It is given with sandalwood paste to stop internal hemorrhage in the stomach (Chatterjee & Pakrashi, 1995).

Cedrus deodar.: Usually prescribed for fever, diarrhea and dysentery. Alcohol bark extract has considerable anti-inflammatory activity against acute and chronic inflammations. The wood possesses diaphoretic, diuretic, and carminative properties and is useful in pulmonary and urinary disorders. The wood powder is one of the ingredients of the well-known Ayurvedic preparations “mondooravataka,” “bhardradarvadi,” and “Taila,” prescribed for anorexia, edema, piles, diabetes, leprosy, and sciatica (Phondke, 1992).

Boerhavia diffusa.: Useful drug for treatment of inflammatory renal diseases and nephrotic syndrome. It is effective for edema and ascites resulting from early cirrhosis of the liver and chronic peritonitis. The plant is reported to be efficacious in abdominal tumors and cancer. It is also credited with antibacterial and cardiotonic properties (Ambasta, 1988a).

Berberis aristata.: The plant is an emmenagogue and is effective in the treatment of jaundice and enlargement of spleen. The fresh berries are laxative, antiscorbutic, and useful in piles, sores, and eye diseases. A decoction is used as a mouthwash for the treatment of gums and toothache (Ambasta, 1988b).

Withania somnifera.: The leaves and fruits are prescribed as an antipyretic and anthelmintic. It is effective for amelioration of painful swelling, scabies, carbuncles, dropsy, cough, hiccup, leukorrhea, menstrual troubles, and rheumatism. It is found beneficial in restoring memory, used for nervous exhaustion, spermatorrhea, impotency, or seminal debility. The decoction of leaves and roots boiled with milk promotes nutrition. It is one of the important ingredients in various Ayurvedic formulations such as “ashwagandha ghrita,” “ashwagandha-arista,” and “narayana taila” (Chatterjee & Pakrashi, 1995b).

Pongamia glabra.: Flowers are prescribed in diabetes. Seed powder is efficacious in whooping and irritating coughs in children. Oil is prescribed internally in stomachic and dyspepsia with sluggish fever; applied externally, either alone or in combination, for skin diseases, in sores, scabies, herpes, and eczema. Leaf juice is effective in cough, flatulence, and diarrhea. Leaf paste is applied internally to ulcers infested with worms. Bark paste has applications in bleeding piles, and decoction is given in beri-beri. Root extract is useful for external application to destroy maggots in foul ulcers and fistulous sores, clearing teeth, and strengthening gums (Chatterjee & Pakrashi, 1992a).

Petrocarpus santalinus.: The decoction of wood is effective as a tonic, astringent, and beneficial in fever, dysentery, and hemorrhage. Wood paste is externally applied in headache, inflammations, boils, piles, and in opthalmia (Chatterjee & Pakrashi, 1992b).

Tinospora cordifolia.: Stem is a constituent of several Ayurvedic formulations used in general debility, dyspepsia, fevers, and urinary diseases. Dry twigs with intact bark are prescribed as anti-inflammatory, antispasmodic, and antipyretic. The roots are a powerful emetic and used for visceral obstruction. The watery extract is recommended for treatment of leprosy. Pulverized fruits are given as a tonic and also for amelioration of jaundice and rheumatism (Ambasta, 1992).

Acrous calamus.: The rhizome constitutes the drug “Calamus” of commerce. In the Ayurvedic system of medicine, the rhizomes are considered to possess anti spasmodic, carminative, and anthelmintic actions. It is also recommended for treatment of epilepsy, amelioration of mental ailments, chronic diarrhea, dysentery, bronchial catarrh, intermittent fevers, glandular/abdominal tumors, kidney/liver troubles, rheumatism, and eczema (Chadha, 1985).

In the past couple of decades, despite the increasing interest of the public in phytomedicine, very few drugs from higher plants have attained any prominence in conventional medical practices. Some reasons for this may be due to lack of standardization and proper scientific validation of herbal drugs (Gilani & Atta-ur-Rahman, 2005).

Considering the high therapeutic amplitude of antioxidants, an urgent need of standardization of botanicals and the pleuropotent medicinal importance of selected plants prompted us to design the current studies on evaluation of an antioxidant and possible anti-inflammatory potential of selected medicinal plants that are usually prescribed in the Indian traditional system of medicine.

Materials and Methods

The samples of the selected plants were purchased from Yogesh Pharmacy (Nanded, India). DPPH and β.-glucuronidase (EC 3.2.1.31, 25,000 units, source: E. coli.) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA), p.-nitrophenyl-β.-D-glucopyranosiduric acid was purchased from CALBIOCHEM (EMD Biosciences Inc. La Jolla, CA, USA), and trypsin was obtained from SISCO Research Lab. Ltd. (Mumbai, India). L-DOPA (3,4-dihydroxyphenyl L-alanine) and glutathione (reduced form) were purchased from S.D. Fine Chemicals Ltd. (Mumbai, India).

All other reagents and solvents used were obtained from commercial sources and were of analytical grade.

Preparation of 50% alcohol extract of selected medicinal plants

The 50% alcohol extracts of selected plants were prepared by extracting approximately 5 g of powdered samples in 50% (v/v) ethanol using a Soxhlet extraction apparatus. The extracts were air-dried and maintained at 4°C for further studies.

Evaluation of DPPH radical scavenging activity

The DPPH radical (DPPH^˙) scavenging assay was carried out according to a method reported by Kato et al. (1988). The DPPH radical scavenging activity of individual plant extract was determined by mixing an equal volume of individual plant extract and DPPH radical (10⁻⁴ M in absolute ethanol) solution. After 20 min reaction time, the absorbance at 517 nm was recorded with a UV-Visible spectrophotometer. The o.d. of the control was considered as a stable DPPH, and IC₅₀ values were determined as the concentration of the individual plant extracts required to reduce 50% of the DPPH free radicals. Glutathione (IC₅₀, 0.45 mg/ml) was used as a reference compound for comparative study.

Determination of OH radical scavenging activity

Hydroxyl radicals scavenging activity assay was performed as per the method described elsewhere (Christos & Dimitra, 2003). The hydroxyl radicals were generated by Fe³⁺/ascorbic acid system. The detection of OH radicals (OH^˙) was carried out by measuring the formaldehyde produced from the oxidation of dimethyl sulfoxide (DMSO). The reaction mixture contained 0.1 mM EDTA, 167 μM Fe³⁺, 33 mM DMSO in phosphate buffer of 50 mM, pH 7.4, and 0.1 ml individual plant extract and 150 μl of ascorbic acid (10 mM in phosphate buffer) were added to start the reaction. The reaction was terminated after 30 min incubation by adding 17% (w/v) CCl₃COOH. The formaldehyde produced was detected spectrophotomertically at 412 nm. The IC₅₀ values were determined as the concentration of the individual plant samples required to achieve 50% of formaldehyde production as compared with control (without sample). Coumarin (IC₅₀, 1.1 mg/ml) was used as a standard drug.

Estimation of vitamin C content

The individual samples of the selected plants were initially extracted in 4% (w/v) oxalic acid. The estimation of vitamin C (L-ascorbic acid) was carried out as per the method described by Sadasivam and Manickam (1996). This method utilizes 2,6-dichlorophenol indophenol, a blue-colored dye that is reduced to a colorless leuco-base by L-ascorbic acid. The amount of vitamin C was calculated as mg/100 g of individual plant sample.

Estimation of total polyphenols

The total polyphenols concentration in alcohol extracts of the selected medicinal plants was carried out by employing the method of Bray and Thorpe (1954). The principle of this method is that phenols react with an oxidizing agent phosphomolybdate (Folin-Ciocalteau reagent), which results in the formation of a blue-colored complex having maximum absorbance at 660 nm. Catechol (500 μg/ml) was used as a standard phenol for construction of a standard curve. The concentration of polyphenols was expressed in terms of mg/g of samples.

PPO inhibition activity

A semipure preparation of PPO was used to realize the effect of individual plant extract on PPO activity. The enzyme PPO was extracted from apple by using the experimental protocol described by Gacche et al. (2003). The PPO assay was performed as per the method described elsewhere (Pathak et al., 1992). The reaction mixture contained L-DOPA (1 ml, 2 mM), 0.5 ml enzyme, 1 ml individual plant extract, and citrate buffer (0.5 ml, pH 4.8, 0.1 M). After 5 min reaction time, the contents of the reaction mixture were observed spectrophotometrically at 470 nm. The activity of PPO in the absence of inhibitor was considered as 100%. The concentration of each test sample giving 50% inhibition (IC₅₀) was then calculated. L-Cysteine (IC₅₀, 0.63 mg/ml) was used as a reference compound.

Trypsin inhibition assay

The trypsin inhibition assay was carried out to understand the antiproteolytic potential of alcohol extract of selected medicinal plants by employing a method described by Tandon et al. (1982). The individual plant extract (0.1 ml) was added with trypsin (0.075 mg/ml) for 20 min. After this incubation time, the substrate, bovine serum albumin (BSA; 6 g/100 ml, in 0.1 M phosphate buffer, pH 7.6) was added. The reaction mixture was incubated for 20 min at 37°C. The reaction was terminated by the addition 3 ml of CCl₃COOH (5%, w/v). The contents of the reaction mixtures were centrifuged at 5000 rpm for 10 min and the acid-soluble fraction obtained in the supernatant was determined spectrophotometrically at 660 nm (Lowry et al., 1951). The activity of trypsin in the absence of inhibitor was considered as 100%. The concentration of each test sample giving 50% inhibition (IC₅₀) was then calculated. For comparative study, salicylic acid (IC₅₀, 0.05 mg/ml) was used as a standard drug.

β.-Glucuronidase inhibition assay

By utilizing the method of Demetrio et al. (1998), the β.-glucuronidase inhibition assay was conducted by preincubating the individual plant extract (0.1 ml) in 0.1 M acetate buffer, pH 7.4, for 5 min at 37°C with 0.8 ml of 2.5 mM p.-nitrophenyl-β.-D-glucopyranosiduronic acid. After this, 0.1 ml of β.-glucuronidase was added. After 30 min incubation, the reaction was terminated by addition of 2 ml of 0.5 N NaOH and the contents were observed spectrophotometrically at 410 nm. The activity of β.-glucuronidase in the absence of inhibitor was considered as 100%. The concentration of each test sample giving 50% inhibition (IC₅₀) was then calculated. Salicylic acid (IC₅₀, 0.052 mg/ml) was used as a standard drug.

Results and Discussion

As summarized in Table 1, the alcohol extracts of all tested plants were found to interact with the stable free radical DPPH, which indicates their radical scavenging ability. The overall range of IC₅₀ values for DPPH radical scavenging activity is between 0.083 and 0.56 (mg/ml). The extract of Acrous calamus. (IC₅₀ 0.083 mg/ml) was found to be the most effective DPPH scavenger.

Table 1. Profile of percent DPPH and OH radical scavenging activity of alcohol extract of selected plants.

Sr. no.	Name of the plant	DPPH IC50 (mg/ml)	OH IC50 (mg/ml)
1	Swertia chirayita.	0.42	NR
2	Cedrus deodar.	0.12	NR
3	Boerhavia diffusa.	0.21	NR
4	Berberis aristata.	0.15	NR
5	Withania somnifera.	0.42	NR
6	Pongamia glabra.	0.56	1.9
7	Petrocarpus santalinus.	0.52	1.6
8	Tinospora cordifolia.	0.22	1.5
9	Acrous calamus.	0.083	1.3
10	Glutathione	0.45	ND
11	Coumarin	ND	1.1

The results presented are the mean values of duplicate measurements.

NR, no results under experimental conditions; ND, not determined.

The DPPH radical scavenging assay has often been used to estimate the antiradical activity of antioxidants. Because of its odd electron, DPPH gives strong absorption band at 517 nm (Gulgun, 2004). The compounds (hydrogen donors) that interact with DPPH radicals reduce the radicals to corresponding hydrazines (Contreas-Guzman, 1982). Moreover, compounds having significant reducing potential can be considered as potential candidates for designing an anti-inflammatory drug that may inhibit the enzyme cyclooxygenase: a key enzyme involved in recruiting inflammation process (Demetrios, 1998).

The results of the OH radical scavenging activity of different plant extracts shows that only Pongamia glabra, Petrocarpus santalinus, Tinospora cordifolia., and Acrous calamus. possessed OH radical scavenging activity, whereas all other plant extracts did not show interaction with OH radicals. Again, the alcohol extract of Acrous calamus. (IC₅₀, 1.1 mg/ml) was graded as the most effective toward scavenging of OH radicals. Pongamia glabra, Petrocarpus santalinus, Tinospora cordifolia. showed OH^˙ scavenging activity in a range of IC₅₀ values of 1.5–1.9 mg/ml.

The OH radicals have been implicated to play a critical role in the physiological control of cell function (Drouge, 2002). The OH radicals react with extremely high rate constants, indiscriminately, with almost every type of molecule found in living cells: sugars, amino acids, phospholipids, DNA bases, organic acids, and may change normal physiological functions of the cells (Barry & John, 1984). Moreover, in rheumatic arthritis and related disorders, the reaction of nitric oxide with superoxide generates peroxynitrite, which, under acid conditions often found in regions of inflammation and ischemia, yields hydroxyl radicals (Brown & Hall, 1992). The hydroxyl radicals thus generated in the above reaction are believed to contribute to membrane damage of the cells in the region of inflammation (Darligton & Stone, 2001).

The redox active antioxidants like ascorbate, glutathione, urate, flavonoids, tocopherols, carotenoids, and hydroxycinnamic acids present in plants (Eastwood, 1999) may be the contributing factors for scavenging of DPPH and OH radicals.

The data presented in Table 2 shows the concentration of vitamin C (ascorbic acid) and total polyphenols in the selected plants. The extract of Petrocarpus santalinus. (45.96 mg/100 g) was found to contain the highest concentration of vitamin C, and the extract of Cedrus deodar. (73.50 mg/g) was found to possess the highest concentration of total polyphenols. All other plants under study were found to possess the considerable amounts of vitamin C and total polyphenols.

Table 2. Summary of vitamin C and polyphenol contents of alcohol extract of selected plants.

Sr. no.	Name of the plant	Vitamin C (mg/100 g)	Phenolics (mg/g)
1	Swertia chirayita.	13.32	45.20
2	Cedrus deodar.	21.72	73.50
3	Boerhavia diffusa.	22.96	42.50
4	Berberis aristata.	26.64	21.50
5	Withania somnifera.	17.37	22.56
6	Pongamia glabra.	26.65	43.05
7	Petrocarpus santalinus.	45.96	39.60
8	Tinospora cordifolia.	30.40	41.95
9	Acrous calamus.	34.43	46.40

The results summarized are the mean values of duplicate measurements.

Ascorbic acid is a reputed antioxidant having an ability to scavenge a wide range of reactive oxygen species, like superoxide anion, singlet oxygen, and H₂O₂, and acts as a chain-breaking antioxidant (Beyer, 1994). The importance of vitamin C as an antioxidant is indispensable.

Plant polyphenols, a diverse group of phenolic compounds (flavonols, flavanols, anthocyanins, phenolic acids, etc.) possess an ideal structural chemistry for free radical scavenging activity, and they have been shown to be more effective antioxidants in vitro. than tocopherols and ascorbate. Antioxidative properties of polyphenols arise from their high reactivity as hydrogen or electron donors from the ability of the polyphenol-derived radical to stabilize and delocalize the unpaired electron (chain-breaking function), and from their potential to chelate metal ions (termination of the Fenton reaction) (Rice-Evans et al., 1997).

The results summarized in Table 3 clearly indicate that, except for Pongamia glabra., all other plant extracts were found to be effective inhibitors of PPO. The alcohol extracts of Tinospora cordifolia. (IC₅₀, 0.2 mg/ml), followed by Swertia chirayita. (IC₅₀, 0.35 mg/ml) and Withania somnifera. (IC₅₀, 0.36 mg/ml) demonstrated a maximum PPO inhibitory effect as compared with other plant extracts, which had a range of IC₅₀ values from 0.67 to 0.96 mg/ml.

Table 3. Inhibitory effect of alcohol extract of selected plants on the activity of PPO, β.-glucuronidase, and trypsin-induced hydrolysis of BSA.

		IC50 (mg/ml)
Sr. no.	Name of the plant	PPO	β.-Glucuronidase	Trypsin
1	Swertia chirayita.	0.35	NR	0.2
2	Cedrus deodar.	0.67	0.22	0.133
3	Boerhavia diffusa.	0.95	NR	0.35
4	Berberis aristata.	0.70	NR	0.45
5	Withania somnifera.	0.36	NR	1.3
6	Pongamia glabra.	NR	NR	0.066
7	Petrocarpus santalinus.	0.65	NR	0.4
8	Tinospora cordifolia.	0.20	NR	1.2
9	Acrous calamus.	0.96	0.067	0.5
10	L-Cysteine	0.63	ND	ND
11	Salicylic acid	ND	0.052	0.05

The results shown here are the mean values of duplicate measurements.

PPO, polyphenol oxidase; BSA, bovine serum albumin; NR, no results under experimental conditions; ND, not determined.

Enzyme PPO (EC 1.14.18.1), a copper-containing enzyme reported to occur in all plants, has been studied as a model oxidizing enzyme as it contains metal ion and utilizes molecular oxygen. By utilizing molecular oxygen, PPO catalyzes the oxidation of a variety of phenols to reactive ortho.-quinones (Timothy et al., 1991). O₂ supplied at concentrations greater than those in normal air has long been known to damage plants, animals, and aerobic bacteria such as Escherichia coli.. Moreover, transition metal–mediated generation of free radicals from oxygen contributes to the development of many diseases like iron overload, rheumatoid arthritis, and cancer (Barry & John, 1984). In general, a system utilizing molecular oxygen and transition metal ions may generate free radicals and manifest altered physiological effects. A strategy for inhibition of this system may minimize the toxic effects of oxygen. In the current study, the inhibition of PPO may relate to the presence of thiol compounds, ascorbic acid, and/or phenolic acids (having metal chelation activity), which are reported as PPO inhibitors (Gacche et al., 2004).

The results of the inhibition of trypsin-induced hydrolysis of BSA using alcohol extracts of selected plants (presented in Table 3) shows that except for the poor antiproteolytic activity of Withania somnifera. (IC₅₀, 1.3 mg/ml), all other plant extracts demonstrated moderate antiproteolytic potential with IC₅₀ values in the range of 0.066–0.2 mg/ml. The extract of Pongamia glabra. (IC₅₀, 0.066 mg/ml) manifested the highest antiproteolytic effect.

A vast body of knowledge elucidating the role of protease inhibition and anti-inflammatory effects has accumulated in recent years. In all of the inflammatory cascades found in the body, there are enzyme-mediated parts that involve serine proteases. As examples, these include the pancreatic enzymes, trypsin, elastase, and enzymes of the complement system. Serine protease inhibition has been considered as one of the targets for designing anti-inflammatory agents (Bilfinger & George, 2002).

It is clear from the results summarized in Table 3 that, except the extracts of Cedrus deodar. (IC₅₀, 0.22 mg/ml) and Acrous calamus. (IC₅₀, 0.067 mg/ml), none of the plant extracts under study showed β.-glucuronidase inhibitory activity. The polymorphonuclear neutrophils (one of the granulocytes) have been implicated as mediators for inducing inflammation. The enzyme β.-glucuronidase is present in the lysosomes of neutrophils and has been reported as one of the mediators for recruiting the inflammation process (Ito et al., 1982; Savill et al., 1995).

Flavonoids, besides their powerful free radical scavenging activity, have been described as anti-inflammatory agents. This group of phenolic compounds has been reported to interact with various enzymes of inflammation including proteolytic enzymes of neutrophils. Furthermore, some of the anti-inflammatory effects may be a result of a combination of radical scavenging and an interaction with enzymes of inflammation (Middleton, 1998; Middleton & Kandaswami, 1992). The presence of specific flavonoid compounds in the plant extracts might be the probable cause for antiproteolytic activity and β.-glucuronidase inhibition.

Conclusions

Herbal preparations have gained popularity among rural and urban communities in India, with a belief that natural resources are safe. However, the fact is that therapeutic approaches with herbal medicine lack information regarding safety and efficacy along with well-organized and rigorous clinical trial evidence. To advocate the scientific merit of herbal medicine, there is an urgent need to introduce standardization and proper scientific validation. Furthermore, the renewed interest in medicinal plants has focused on herbal cures among indigenous populations around the world. The results of the current study may supplement and strengthen the process of standardization and validation of herbal drugs containing active ingredients.

Acknowledgments

The authors are thankful to UGC India for financial assistance and HOD School of Life Sciences, S. R. T. M. University Nanded (MS) India, for providing the necessary facilities during this work.