MEDICINAL VALUE AND CLINICAL SIGNIFICANCE OF CENTELLA ASIATICA

Author : Palak R 1 , Neeleshwari Parmar2 * , Kirtan Gupta3, Pankaj Kushwah4

1243Chameli Devi Institute of Pharmacy, Indore.

CORRESPONDING AUTHOR

Neeleshwari Parmar

Email : parmarneeleshwari@gmail.com

ABSTRACT

Plant-based drug discovery has drawn the attention of researchers, especially the one used as traditional medicines. Centella asiatica is traditional Ayurveda medicine widely used in India and across Asia for treating a variety of diseases. The aerial parts and roots are used for medicinal purpose, and its chemical constituents have wide therapeutic applications in areas of antimicrobial, anti-inflammatory, anticancer, neuroprotective, antioxidant, and wound healing activities. Many of its uses have been proven scientifically, and bioactive ingredients have been validated. In this review, we have done a critical evaluation of available literature looking for the pharmacological importance of C. asiatica. Further studies will be helpful to discover more bioactive compounds their exact mode of actions.

INTRODUCTION

The plant is commonly known as Gotu Kola, Asiatic pennywort, Indian pennywort or Spadeleaf and belongs to Umbelliferae/Apiaceae family. In China, Southeast Asia, India, Sri Lanka, Oceania, and Africa, the plant has long been used as vegetable. In Southeast Asia, it is traditionally used for the treatment of a wide variety of disorders such as skin diseases, rheumatism, inflammation, syphilis, mental illness, epilepsy, hysteria, dehydration, and diarrhea. Centella asiatica (Gotu Kola) is used in Indian systems of medicine for enhancing memory and for the treatment of skin diseases and nervine disorders. The plant medicinal properties have long been utilized by the people of Java and Indonesia. In China, it is indigenously called as Gotu kola, and over 2000 years ago, it was one of the documented “miracle elixirs of life”. Herbal medicines can be used as adaptogens, these plant derived drugs either reduce stress reactions in the alarm phase and provide a certain degree of safety against long-term stress. C. asiatica (Umbelliferae) syn. Hydrocotyle asiatica is used to treat various ailments across India which includes body aches, headaches, insanity, asthma, leprosy, ulcers, eczemas, and wound healing . To find out new potential compounds for therapeutic use, screening of medicinal plant is vital.

Gotu Kola, also known as Centella Asiatica, is a tiny, perennial herbaceous plant that has been used for a long time in Asian traditional medicine systems, especially in Ayurveda, Traditional Chinese Medicine

(TCM), and Indonesian folk medicines. It has been valued for ages due to its capacity to improve general health, encourage the healing of wounds, and improve cognitive function.

The pharmacological properties of the plant are mostly attributed to its rich composition of bioactive chemicals, which include asiaticoside, madecassoside, and asiatic acid. It has been demonstrated that these substances have neuroprotective, anti-inflammatory, antioxidant, and skin-regenerating qualities. Centella Asiatica is becoming more and more acknowledged in modern medicine, especially in the fields of neurobiology and dermatology, as scientific research on the plant's potential for medicinal use continues. The objectives of this review are to investigate Centella Asiatica's historical uses, assess the available scientific data, and talk about possible future uses in contemporary therapeutic settings.

CHEMICAL CONSTITUENTS

The plant has long been used as folklore medicine for the treatment of a variety of diseases. Chemically been identified leading to therapeutic properties. Asiatic acid, asiaticoside, and madecassoside form the major constituents responsible for pharmacological value apart from being rich in flavonoids and terpenoids. Centelloid was term given for different constituents of secondary metabolites produced by plant which mainly comprised of pentacyclic triterpenoid saponins. P-cymene-(44%) along with other volatile compounds was found to be in a prominent amount in the essential oil of C. asiatica on analysis with gas chromatography-mass spectrometry (GC-MS). Centellin, asiatic, and centellicin were isolated from the aerial part of the plant, and further, their structures had been determined using 2D nuclear magnetic resonance technique. From plant extractusing high-performance liquid chromatography to identify bioactive compounds, madecassoside, asiaticoside, madecassic acid, and asiatic acid were found in the significant amount. A quantitative estimation of triterpenoids showed highest asiaticoside content (6.42%) in leaf samples collected in Mangoro region. New triterpene and a saponin, 2α,3β,23- trihydroxyurs-20-en-28-oic acid and 2α,3β,23 trihydroxyurs-20-en-28-oic acid O-α-l-rhamnopyranosy- (1→4)-O-β-d glucopyranosyl (1→6)-O-β-d glucopyranosyl ester, have been isolated from the aerial part of C. asiatica, and their structures were determined using spectral methods.

Phytochemical Composition and Mechanisms of action

Centella Asiatica's extensive phytochemical composition accounts for a wide spectrum of its outstanding effects. The plant's medicinal qualities are attributed to a number of bioactive substances, including amino acids, polyphenols, triterpenoids, flavonoids, and phytosterols. These substances combine to offer the skin a number of advantages.

Triterpenoids

Perhaps the most researched and well-known components of Centella Asiatica are its triterpenoids. Asiaticoside, madecassoside, asiatic acid, and madecassic acid are among the chemicals that provide the herb its skin-healing qualities.

1. Asiaticoside: Research has demonstrated that the glycoside Asiaticoside enhances wound healing and stimulates the creation of collagen. It increases the skin's tensile strength, which lessens the likelihood of scarring and helps stop wounds from healing again. Due to its anti-inflammatory qualities, astaxicoside is also useful in lowering skin irritation and redness.

2. Madecassoside: Another important triterpenoid with strong antioxidant and anti-inflammatory properties is madecassoside. It relieves skin irritation, lessens inflammation, and guards against oxidative stress brought on by free radicals. Because it relieves irritation and encourages healing, madecassoside is especially helpful for sensitive and acne-prone skin.

3. Madecassic Acid and Asiatic acid: It has been demonstrated that the triterpenic acids: madecassic and Asiatic acid increase the synthesis of collagen and increase the suppleness of the skin. Additionally, they

have anti-inflammatory and wound-healing properties, which makes them useful in the treatment of a variety of skin issues, such as eczema, acne, and scarring.

Flavonoids and polyphenols

Flavonoids and polyphenols, which are potent antioxidants that help shield the skin from harm from the environment, are abundant in Centella Asiatica. Free radicals are unstable chemicals that have the potential to harm cells and hasten the aging process. These substances counteract them.

1. Antioxidant protection: Centella Asiatica's flavonoids and polyphenols aid in shielding the skin from oxidative stress brought on by pollution, UV rays, and other external variables. Preventing early aging, which includes the development of fine lines, wrinkles, and hyperpigmentation, requires this protection.

2. Anti-Inflammatory actions: Flavonoids and polyphenols have anti-inflammatory actions in addition to their antioxidant qualities. Centella Asiatica is a wonderful option for treating inflammatory skin diseases like rosacea, eczema, and acne since they assist to reduce redness and inflammation.

Amino acids

Amino acids are found in Centella Asiatica and are the building blocks of proteins. These substances are essential for preserving the skin's general health, barrier integrity, and moisture.

1. Hydration: By boosting the skin's natural moisturizing factors (NMFs), amino acids assist the skin hold onto moisture. Improved skin hydration, elasticity, and texture are the outcomes of this.

2. Barrier function: The skin's barrier function, which is crucial for shielding the skin from outside aggressors and halting moisture loss, is supported by amino acids. Additionally, a robust barrier function lowers the chance of sensitivity and discomfort.

Phytosterols

Compounds originating from plants called phytosterols have the ability to soothe and reduce inflammation in the skin. They aid in promoting healing, lowering redness, and restoring the skin's natural barrier.

1. Skin soothing: Centella Asiatica is a good choice for sensitive and reactive skin types since phytosterols aid to soothe inflamed skin and minimize redness.

2. Repairing the Skin's Barrier: Phytosterols help to restore the skin's barrier, which is necessary to keep the skin resilient and healthy. Strong barrier function aids in shielding the skin from outside aggressors and halting moisture loss.

Applications of Centella asiatica in modern skincare

Centella Asiatica's amazing ability to treat a wide range of skin issues has fueled its move from traditional medicine to modern skincare. These days, Centella Asiatica is a common component in many cosmetic products, such as masks, toners, serums, and creams. Dermatologists, formulators, and skincare lovers all love it for its efficacy and adaptability.

Anti-Aging skincare

Centella Asiatica's contribution to anti-aging skincare products is among its most notable features. The growth of wrinkles, fine lines, and sagging skin are the results of a decrease in collagen production in aging skin. It has been demonstrated that the triterpenoids in Centella Asiatica, in particular asiaticoside and madecassoside, stimulate the manufacture of collagen, which is necessary to preserve the firmness and suppleness of the skin.

1. Collagen boosting: Centella Asiatica helps to firm the skin, minimize wrinkles, and improve skin suppleness by encouraging the production of collagen. Because of this, it's a fantastic component for anti- aging serums and creams.

2. Antioxidant protection: One of the main causes of aging is oxidative stress, which is prevented from occurring on the skin by Centella Asiatica's antioxidant qualities. Centella Asiatica prevents premature aging and preserves youthful-looking skin by scavenging free radicals.

3. Skin firming: Using Centella Asiatica-containing products on a regular basis can result in firmer, more robust skin. The herb's increased ability to synthesize collagen and antioxidant defense helps to improve the skin's overall tone and texture.

Soothing and Healing products Due to its well-known calming and restorative qualities, Centella Asiatica is a perfect addition to cosmetic products intended for skin that is damaged, sensitive, or irritated. Centella Asiatica is used as a soothing serum for skin prone to acne or as a calming cream after sun exposure to help reduce inflammation, lessen redness, and hasten the healing process.

1. Wound healing: It has long been known that Centella Asiatica can aid in the healing of wounds. It speeds up the healing process, increases the skin's tensile strength, and promotes the creation of collagen. This makes it useful for treating burns, wounds, and other skin ailments in addition to acne blemishes.

2. Skin barrier repair: Centella Asiatica plays a vital role in repairing and fortifying the skin's barrier function, which is necessary to keep skin robust and healthy. Strong barrier function aids in shielding the skin from outside aggressors and halting moisture loss.

Hydrating and Moisturizing products

Centella Asiatica is a desirable ingredient in moisturizers and hydrating serums since it contains amino acids and other moisturizing compounds. These products aid in restoring the moisture balance of the skin, enhancing its general brightness, and improving its texture.

1. Deep hydration: By boosting the skin's natural moisturizing agents, Centella Asiatica helps to retain moisture in the skin and promotes hydration. This is especially advantageous for skin that is parched or dehydrated.

2. Better skin texture: Using Centella Asiatica on a regular basis can result in softer, smoother skin. Because of its moisturizing qualities, the skin feels more robust and has a better texture.

3. Enhanced radiance: Skin that has received enough moisture looks healthier and more radiant. Centella Asiatica keeps the skin nourished and hydrated, which enhances the skin's natural radiance.

Scar Treatment and Management

Centella Asiatica is a useful ingredient for treating and lessening the appearance of scars because of its ability to heal wounds and increase collagen. Stretch marks, acne scars, and surgical scars can all be gradually faded and the texture of the skin improved with Centella Asiatica.

1. Scar reduction: Centella Asiatica aids in the reduction of visible scars by encouraging the synthesis of collagen and boosting skin regeneration. Additionally, it can make scarred skin seem smoother and more even in texture.

2. Stretch mark prevention: Products meant to lessen and prevent stretch marks frequently contain Centella Asiatica. rapid growth or weight fluctuations because of its capacity to increase skin elasticity and stimulate the creation of collagen.

3. Post-Surgical treatment: To promote healing and reduce scarring, Centella Asiatica is often advised for post-surgical treatment. Because of its capacity to promote wound healing and skin regeneration, it is an important component of post-operative skincare products.

Acne treatment

Acne is a prevalent skin ailment that impacts millions of individuals globally. Centella Asiatica is a useful ingredient for treating acne and averting recurrences due to its anti inflammatory, antibacterial, and restorative qualities.

1. Effects on inflammation:

   Centella Asiatica reduces acne-related inflammation, which can lessen the size and redness of pimples. Additionally, its calming qualities lessen the chance of post-inflammatory hyperpigmentation and soothe inflamed skin.

2. Antibacterial properties:

   Centella Asiatica has antibacterial properties that help stop the growth of bacteria that cause acne, which lowers the chance of breakouts. Because of this, it's a useful component in cleansers, toners, and spot treatments for acne.

3. Healing and Recovery:

Centella Asiatica helps acne lesions to heal, lowering the chance of scarring and enhancing the skin's overall appearance. As a result of its capacity to increase collagen synthesis, acne scars gradually disappear and skin texture improves.

Scientific evidence supporting the efficacy of centella asiatica

A significant amount of scientific study backs up Centella Asiatica's rising appeal in the cosmetics sector. Extensive research has examined the constituents of Centella Asiatica and their impact on the skin, offering strong proof of its effectiveness.

1. Wound Healing and Skin regeneration

   The capacity of Centella Asiatica to accelerate skin regeneration and facilitate wound healing is one of its most well-established advantages. Asiaticoside, one of the main triterpenoids found in Centella Asiatica, has been demonstrated in numerous studies to promote collagen synthesis, a necessary process for wound healing.

2. Collagen synthesis:

   Asiaticoside dramatically enhanced the synthesis of collagen in fibroblast cells, which are in charge of generating collagen in the skin. Additionally, the study discovered that asiaticoside increased the tensile strength of freshly produced skin, strengthening it against harm and reducing the likelihood of scarring.

3. Wound healing:

In a different study, Centella Asiatica extract was shown to hasten wound healing in animal models. According to the study, Centella Asiatica improved the quality of the healed skin, decreased inflammation, and accelerated the rate of wound healing. These results validate Centella Asiatica's historic use in the treatment of wounds and other skin ailments.

Anti-Inflammatory and Antioxidant effects

Numerous studies have been conducted on the antioxidant and anti-inflammatory qualities of Centella Asiatica. These characteristics are essential for lowering inflammation, shielding the skin from the elements, and delaying the signs of premature aging.

1. Effects on inflammation:

   Using animal models, a study published in "Phytomedicine" examined the anti-inflammatory properties of Centella Asiatica extract. According to the study, Centella Asiatica greatly decreased inflammation and stopped pro-inflammatory cytokine production. These results imply that Centella Asiatica may be useful in the treatment of inflammatory skin diseases such rosacea, eczema, and acne.

2. Antioxidant protection:

   An additional investigation into Centella Asiatica's antioxidant qualities was published in the "Journal of Ethnopharmacology". Strong free radical scavenging activity of Centella Asiatica extract was identified in the study, which helps shield the skin from oxidative stress. Maintaining healthy, youthful-looking skin and preventing premature aging depend on this antioxidant protection.

3. Anti-Aging and Skin firming

   Numerous research has investigated Centella Asiatica's possible anti-aging benefits, with encouraging outcomes. The herb is a useful component in anti-aging skincare products because of its capacity to increase collagen formation, enhance skin suppleness, and guard against oxidative stress.

4. Better skin elasticity:

   The benefits of a cream containing Centella Asiatica extract on skin elasticity and firmness were examined in a clinical trial that was published in the "International Journal of Cosmetic Science". The elasticity and firmness of the subjects' skin significantly improved after eight weeks of use, according to the study. These findings demonstrate Centella Asiatica's ability to improve overall skin texture and lessen the visibility of fine lines and wrinkles.

5. Decrease in wrinkles:

An additional investigation on the impact of Centella Asiatica extract on aging mice's skin was published in "Phytotherapy Research". According to the study, Centella Asiatica greatly enhanced skin moisture and decreased the appearance of wrinkles. The researchers came to the conclusion that Centella Asiatica is a useful component for treating and preventing the symptoms of aging because of its collagen boosting and antioxidant qualities.

Scar Treatment and Management

Numerous research has examined the potential of Centella Asiatica to enhance skin texture and lessen the appearance of scars in the treatment of scars.

1. Scar reduction:

   The effects of a cream containing Centella Asiatica extract on hypertrophic scars were studied in a clinical experiment that was published in the "Journal of Cutaneous and Aesthetic Surgery". According to the study, compared to those who used a placebo, individuals who used the cream had significantly better skin texture and smaller scars. The scientists came to the conclusion that Centella Asiatica works well to improve the general condition of the skin and lessen the appearance of scars.

2. Prevention of stretch marks:

A further study that was published in "Advances in Dermatology and Allergology" looked at how a lotion containing Centella Asiatica affected pregnant women's stretch marks. According to the study, stretch marks were less common in women who used the cream than in those who received a placebo. The capacity of Centella Asiatica to increase skin elasticity and stimulate the formation of collagen is how the researchers explained these outcomes.

Centella asiatica in combination with other ingredients

Centella Asiatica is a common ingredient in cosmetics since it works well with other active compounds. To maximize Centella Asiatica's benefits and produce formulations that work well together, formulators frequently blend it with other botanicals and manmade substances.

Centella Asiatica and Niacinamide

Vitamin B3, or niacinamide, is a multipurpose ingredient in skincare products that has anti aging, anti- inflammatory, and brightening effects. Together, these two components help to reduce redness, soothe inflammation, and enhance the texture of the skin when mixed with Centella Asiatica. This combo works very well for treating hyperpigmentation, rosacea, and acne.

1. Enhanced Anti-Inflammatory effects:

   The potent anti-inflammatory properties of Centella Asiatica and niacinamide make it a perfect remedy for inflammatory skin disorders including rosacea and acne. While Centella Asiatica encourages healing and lessens redness, niacinamide also helps to enhance skin texture and minimize the appearance of enlarged pores.

2. Even skin Tone and Brightening:

Niacinamide is well-known for its capacity to even out skin tone, minimize the appearance of dark spots, and lessen hyperpigmentation. It helps to balance out skin tone and improve overall skin radiance when used with Centella Asiatica. Brightening serums and creams that aim to address hyperpigmentation and discolouration frequently contain this combination.

Centella Asiatica and Hyaluronic acid

Strong humectant hyaluronic acid draws and holds moisture in the skin. When used with Centella Asiatica, it promotes skin healing, lessens the appearance of fine lines and wrinkles, and amplifies the moisturizing properties of Centella Asiatica. For skin types that are dry or dehydrated, this combination is perfect.

1. Intensive hydration:

   Centella Asiatica helps to strengthen the skin's barrier function and stop moisture loss, while hyaluronic acid helps to attract moisture into the skin. Deep hydration is provided by this combination, which also keeps the skin supple and plump.

2. Relaxing and Soothing:

Hyaluronic acid's moisturizing qualities together with Centella Asiatica's relaxing qualities make this combo perfect for calming sensitive or irritated skin. It keeps the skin moisturized and nourished while assisting in the reduction of redness and inflammation.

Centella Asiatica and Vitamin C

Strong antioxidant vitamin C makes skin appear brighter and less prone to black spots. When combined with Centella Asiatica, it increases the creation of collagen and offers complete protection against environmental damage. This combo is frequently present in brightening and anti-aging serums.

1. Brightening and Antioxidant defense:

   Centella Asiatica offers extra antioxidant defense while vitamin C helps to lighten the face and minimize the appearance of dark spots. This combination works especially well to prevent and repair wrinkles, fine lines, and hyperpigmentation, which are indications of aging.

2. Collagen boosting:

   Centella Asiatica and vitamin C both increase the formation of collagen, which enhances skin suppleness and minimizes the appearance of wrinkles. For people who want to tighten and firm their skin while also enhancing its general tone and texture, this combination is perfect.

   The impact of centella asiatica on the cosmetic industry

   Centella Asiatica's ascent to prominence in the cosmetics sector has had a profoundly transforming effect. It is a favorite with formulators and customers alike because of its mild, calming qualities and capacity to address a broad spectrum of skin issues.

   Consumer Demand and Market growth

   the demand for skincare products containing Centella Asiatica has increased due to consumers' rising awareness of the benefits of natural and botanical components. Customers are looking for skincare products that are mild, effective, and made of natural ingredients more and more. Centella Asiatica and other botanical components are becoming increasingly popular as a result of this shift in consumer tastes.

   • Expanded product offerings:

     As a result of growing consumer demand, more skincare products now include Centella Asiatica. Nowadays, Centella Asiatica is a widely used ingredient in a variety of products, ranging from cleansers and toners to serums and creams. Centella Asiatica has been the focus of numerous skincare lines created by businesses, demonstrating the plant's adaptability and potency.

   • Global market growth:

     The natural and organic skincare segment has had the most growth in the global skincare market, thanks in part to Centella Asiatica's popularity. There will likely be a further increase in demand for botanical ingredients like Centella Asiatica as people look for solutions that are both eco-friendly and effective.

     Innovation and Research

     The fascination of the cosmetic business with Centella Asiatica has also sparked creativity and investigation. Researchers and formulators are always coming up with new ways to maximize Centella Asiatica's advantages and boost the product's effectiveness in skincare regimens.

   • Novel extraction techniques: More efficient extraction techniques have made it possible for formulators to separate and concentrate Centella Asiatica's active ingredients. As a result, more effective and focused skincare solutions that fully benefit from Centella Asiatica have been developed.

   • Synergistic formulations:

     By combining Centella Asiatica with additional active substances, formulations that work well together can provide greater advantages. Because these formulas target numerous skin issues at once, customers find them more appealing and effective.

   • Clinical research:

More studies examining Centella Asiatica's effects on the skin have been conducted as a result of the growing interest in the plant. The results of this study further support the usage of Centella Asiatica in cosmetics by offering insightful information on the plant's possible advantages and modes of action.

CONCLUSION

Centella Asiatica is evidence of the continued value of traditional medicinal plants in contemporary medicine. An increasing amount of scientific study has validated its numerous medicinal characteristics, which include skin regeneration, wound healing, cognitive improvement, and neuroprotection. Centella Asiatica is a versatile plant whose bioactive chemicals, especially asiaticoside and madecassoside, have demonstrated substantial potential in a range of clinical applications. Centella Asiatica is positioned to become more and more important in therapeutic practices as the merging of traditional knowledge with contemporary scientific investigation proceeds, especially in the fields of neurology, dermatology, and anti-aging medicine. To completely understand its mechanisms of action and maximize its clinical application, more research is needed. We can make sure that Centella Asiatica continues to be a useful tool in the search for safe, all-natural health solutions by investigating and comprehending its many uses.

REFERENCES

1. B. Brinkhaus, M. Lindner, D. Schuppan & E. G. Hahn, “Chemical, pharmacological and clinical profile of Centella asiatica,” Phytomedicine , 2000; 7(5): 427–448.

2. J. T. James & I. A. Dubery, “Pentacyclic triterpenoids from Centella asiatica: A review of biological activities,” Journal of Ethnopharmacology , 2009; 123(3): 297–305.

3. K. J. Gohil, J. A. Patel & A. K. Gajjar, “Pharmacological review on Centella asiatica,” Indian , 2010; 72(5): 546–556.

4. E. Orhan, “Centella asiatica: Neuroprotective potential,” Journal of Ethnopharmacology , 2012; 141(1): 1–13.

5. Shukla, A. M. Rasik & B. N. Dhawan, “Asiaticoside-induced wound healing,” Journal of , 1999; 65(1): 1–11.

6. L. Suguna, P. Sivakumar & G. Chandrakasan, “Dermal wound healing in rats,” Indian Journal of , 1996; 34(12): 1208–1211.

7. N. E. Gray et al., “Neuroprotection by Centella asiatica,” Phytochemistry Reviews , 2018; 17: 161– 194.

8. W. Bylka et al., “Centella asiatica in cosmetology,” Postepy Dermatol Alergol , 2013; 30(1): 46– 49.

9. K. S. Park, “Skin disease mechanisms,” Evidence-Based Complementary and Alternative , 2021.

10. S. C. Tan et al., “Madecassoside in inflammation,” Applied Sciences , 2021; 11.

11. Kandasamy et al., “Antioxidant activity,” Plants , 2023; 12.

12. D.Widgerow et al., “Scar management,” Aesthetic Plastic Surgery , 2000; 24(3): 227–234.

13. D. Bensky et al., Chinese Herbal Medicine , 2004.

14. R. A. Indria et al., “Stretch marks reduction,” Journal of Health Sciences , 2021; 15(3): 196–201.

15. T. K. Lin et al., “Skin barrier repair,” International Journal of Molecular Sciences , 2018; 19.

16. R. H. Singh & R. P. Rastogi, “Role in Ayurveda,” Ancient Science of Life , 1983; 3(2): 79–86.

17. K. G. Mohandas Rao et al., “Memory enhancement,” Phytotherapy Research , 2007; 21(9): 845– 852.

18. Y. Chen et al., “Triterpenes in wound healing,” Phytomedicine , 2003; 10(6–7): 540–546.

19. M. H. Kumar & Y. K. Gupta, “Cognition and oxidative stress,” Journal of Pharmacy and , 2002; 54(3): 345–350.

A. K. Dash et al., “Antimicrobial activity,” Asian Pacific Journal of Tropical Biomedicine , 2011; 1(2): 152–157.