Antimicrobial activity of a Unani eye drop formulation in experimentally induced corneal ulcer in rabbit’s eye

· Volume 6
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*Abdul Latif, MD1; Abdul Razique, MD1 and R. R. Sukul, MS2

1Department of Ilmul Advia, A.K.T. College, 2Institute of Ophthalmology, J.N. Medical College and Hospital, Aligarh Muslim University, Aligarh-202002,  India.

Correspondence to: Abdul Razique – arazik_49@rediffmail.com

Submitted: 16 January 2010

Accepted for publication: 10 August 2010

Abstract

In the present study, the Unani eye drop (UED) formulation was prepared for its beneficial effects in infective conditions of the eyes. The UED was prepared from seven herbo-mineral ingredients. The clinically isolated strain of Staphylococcus aureus was used to detect the in-vitro antimicrobial activity of UED and standard drug (Moxifloxacin hydrochloride eye drop) after that the in-vivo antimicrobial activity of placebo, test drug and standard drug was also carried out on corneal ulcers in rabbit eyes. The in-vitro antimicrobial study showed that UED at a dose of 10 µl/disc showed antimicrobial activity against Staphylococcus aureus. The result of Unani eye drop formulation in the treatment of corneal ulcer was almost comparable to that of Moxifloxacin eye drops and showed significant (p<0.05) antimicrobial activity against control. The study revealed that UED formulation is a potential antimicrobial drug having antimicrobial activity against Staphylococcus aureus bacterial strain.

Key words: Unani eye drop, moxifloxacin, corneal ulcer, antimicrobial, placebo.

* Corresponding author. Dr. Abdul Latif,   Associate Professor & Chairman, Department of Ilmul Advia (Unani Pharmacology & Pharmaceutical Sciences), Faculty of Unani Medicine, Aligarh Muslim University, Aligarh, 202002, India, Telephones: 0091-571-2401007(office), 0091-9411491277 Email: abdullatifamu@gmail.com

Introduction

For thousands of years, natural products have been used in traditional medicine all over the world and predate the introduction of antibiotics and other modern anti-inflammatory drugs. It is estimated that local communities have used about 10% of all flowering plants on earth to treat various infections, although only 1% have gained recognition by modern scientists.1 Plants are rich in a wide variety of secondary metabolites such as tannins, alkaloids and flavonoids, which have been found in-vitro to have antimicrobial properties.2 The use of herbal and mineral drugs in the treatment of various diseases is mentioned by Unani physicians in their classical literature. It is also believed that plant derived drugs are safe and more dependable and have little side effects than the costly synthetic drugs, many of which have adverse effects and beyond the reach of poor patients. Thus emphasis was laid on the plants based drugs as they are cheap, easily available, rich in alkaloids, and possess significant biological activity. The main emphasis for the treatment of inflammatory and infective eye diseases is the topical application of anti-inflammatory drugs and antibiotics. Although very effective, resistance is developing against antibiotics3. The use of herbal and mineral drugs in the treatment of ocular ailments is mentioned by Unani physicians in their classical literature. The practice of using herbal drugs in the treatment of ocular ailments is well documented in Unani literature4 as well as in Ayurvedic literature5.

Although there are many drugs in Unani repository which are mentioned in classical books or used in Unani clinical practice effectively in treatment of eye diseases by various Unani physicians. The efficacy of many traditional herbal medicines in curing diseases is now being gradually recognized in modern medicine as well6,7. There are several reports on the antimicrobial activity of different herbal extracts.8 The WHO recognized Indian system of medicine which included Unani system of medicine. According to the WHO, medicinal plants would be the best source for obtaining a variety of drugs.9 Various research studies have demonstrated the effectiveness of polyherbal formulations in management of acute and chronic conjunctivitis.10

The Unani system of Medicine originated in the Greek but later Arab physicians explored the knowledge of this system to all over the world. They provided a lot of descriptions about ocular ailments, because the ocular diseases are quite common in those places that have unbearable heat, scorching wind and where the air is full of dust and sand particles. They also mentioned reasons for the presence of large number of eye specialists compared to other diseases.11 Alhazen (384-435), the “father of optics”, studied the anatomy of the eye extensively. He made important contributions to ophthalmology and eye surgery and proposed the first correct explanations of the process of sight and visual perception in his Book of Optics which was known as Opticae Thesaurus in Europe.12 Razes (841-926) in his book ‘Al-Hawi’ quotes that it is advised rasaut (extract of Berberis aristata) for topical application once a week in eyes. Rasaut is beneficial in this way in increasing the vision. He also described that instilling of aqueous solution of rasaut for prevention of cataract and diminution of vision.13

The active constituents of eye drop formulation rose petals contain essential oil, aromatic volatile oil, a glycoside quercitrin, gallic acid, resin, malic acid, tannic acid, quercitannic acid and red colouring matter14. Another ingredient Symplocos racemosa contains three alkaloids Loturine-0.24%, Colloturine- 0.02% and Loturidine- 0.06% isolated from the bark.15 The dried rhizomes of Coptis teeta are medicinally important it contains 8-8.5% of berberine that is considered as the active ingredients of the plant. Besides this the rhizome also contains alkaloid like coptine (0.08), coptisine (0.02) and jatrorrhizine (0.01%), palmitine (traces), epiberberine, resin (1.5-2.7%) and ash (3.1-3.3%). 16

In the present study, the Unani eye drop formulation is evaluated for its antimicrobial activity using in-vitro and in-vivo experimental models. The constituents of Unani eye drops are known to possess antimicrobial and anti-inflammatory properties and are used in the traditional medicine for the treatment of a variety of ocular disorders.

Materials and methods

(i) Materials

Blood agar (MV073; Hi media, India), nutrient broth culture media (M002, Hi media Laboratories, Mumbai), Mueller-Hinton agar media (M173; Hi Media, India) moxifloxacin hydrochloride eye drops (Mofelder)supplied by Elder Health Care Ltd. Navi Mumbai, 4% lignocaine hydrochloride (Xylocaine) topical solution USP drops (AstraZeneca Pharma India Ltd., Bangalore), fluorescein sodium ophthalmic strips USP (Fluo) (Contacare Ophthalmics and Diagnosis, Gujarat) and non absorbable suture U.S.P. (Ethicon Johnson & Johnson Ltd, H.P.) were used for study.

 (ii) Unani eye drop formulation

The ingredients of the Unani eye drop formulation include stem wood of Berberis aristata DC., rhizome of Coptis teeta Wall., seeds of Cassia absus Linn., bark of Symplocos racemosa Roxb., flower of Azadirachta indica A. Juss., Alum and aqua distillate of Rosa damascena Mill. Phenyl ethyl alcohol was used as preservative. Specimen having voucher No. (Berberis aristata SC-0111/09), (Coptis teeta SC-0110/09), (Cassia absus SC-0108/09),  (Symplocos racemosa SC-0109/09), (Azadirachta indica SC-0112/09), (Alum SC-0113/09) and (aqua distillate of Rosa damascene SC- 0107/09) are preserved in the museum at  Department of Ilmul Advia, Ajmal Khan Tibbia College, Aligarh Muslim University, Aligarh, India.

(iii) Preparation of Unani eye drop formulation

The Unani eye drops were prepared under aseptic conditions with slight modification as per the method described by Ghulam.17 Berberis aristata DC. (stem wood) 140 gm, Coptis teeta Wall. (rhizome) 24 gm, Cassia absus Linn. (seed) 20 gm, Symplocos racemosa Roxb. (bark) 24 gm, Azadirachta indica A. Juss. (flower) 28 gm were coarsely powdered and soaked in one liter Arq-e-gulab (distillate of Rosa damascena) for 24 hours in a round bottom flask. The mixture was heated at 40-50 °C gently for three hours. The mixture was then cooled, filtered and 4 gm Alum was dissolved in the filtrate. The resulting solution was again filtered and made up to one liter with Arq-e-gulab. 5 ml Phenyl ethyl alcohol was added as preservative in the solution. Finally the solution was filtered through syringe filter (sterile cellulose acetate 25 mm/0.2μm) and then stored in sterilized vials (sterilized by autoclave).

Antimicrobial activity

(i) Bacterial strains

Clinical isolates of Staphylococcus aureus was obtained from the Interdisciplinary unit of Biotechnology, Aligarh Muslim University, Aligarh.

(ii) Inoculam preparation

The antimicrobial activity of Unani eye drop formulation was evaluated by disc diffusion methods against Staphylococcus aureus as described by Baur et al. 18 The bacterial strain was cultured on blood agar culture media and incubated at 37 °C for 18 hours, after this a single colony of bacteria was subcultured on nutrient broth and incubated for 18 hours. The culture suspensions were prepared and adjusted to approximately 105 c.f.u. of bacteria/ml. Five hundred microliters of the inoculam were spread over plates containing sterile Mueller–Hinton agar and paper filter discs (6 mm) impregnated with an eye drop of 10 µl/disc and standard drug placing them on culture plates, already seeded with bacterial strains. The plates were left for 30 min at room temperature to allow the diffusion of the extract and incubated at 37 °C for 18-48 hours. The degree of sensitivity was measured by easily visible areas of zone of inhibition from the disc into the surrounding medium.

(iii) Animal model

Albino rabbits of either sex weighing 1.5-2 kg were used for experimentation. The animals were provided food and tap water ad libitum. The animals were maintained at constant room temperature (27.0 ± 1.0 ­­°C), humidity 65-75% and 12 hours light/12 hours dark cycle. This study was performed as per guidelines and norms of ethics. Permission and approval for animal studies were obtained from Jawaharlal Nehru Medical College and Hospital Animal Ethics Committee, Aligarh Muslim University, Aligarh, India.

(iv) Experimental production of corneal ulcer in rabbit’s eye

Before any experimental procedure, Moxifloxacin hydrochloride eye drops was instilled into both eyes of all rabbits for two days and conjunctival sac was tested for sterility by culture after two days.

The ulcer was produced as the technique described by Khan.19 Lids were sutured by non absorbable suture for three days. On the fourth day, the sutures were cut, eyes were opened and conjunctival smear was taken from the site of the ulcer and inoculated on blood agar plates to confirm the presence of S. aureus in each eye. The corneal ulcer into rabbit’s eye was confirmed with fluorescence strips. The animals were divided into three groups, each group comprised of six rabbits. Distilled water was instilled to Group I rabbits to serve as a control. Rabbits in Groups II and III were instilled with 0.1 ml of Unani eye drops and Moxifloxacin eye drops respectively for 6 days. All groups were administered with test drug, standard and placebo at every two hours throughout the experiment. Signs of corneal ulcer were scored on day 1, 2, 3, 4 and day 5 after respective assigned treatment.

(v) Clinical examination

The eyes of all 18 rabbits were examined daily and the clinical findings were recorded as described by Hogan et al.20. Corneal ulcers were examined daily and conjunctival swabs were obtained daily for culture of bacteria.

Statistical analysis

The data was statistically analyzed using one-way analysis of variance (ANOVA) method. The minimum level of significance was fixed at p < 0.05.

Results

In this study, we tested Unani eye drop formulation and a drug standard (moxifloxacin hydrochloride eye drop 0.5% w/v) at a dose of 10 µl/disc for antimicrobial activity against Staphylococcus aureus. In-vitro analysis with the eye drops and standard drug showed zone of inhibition of 12mm and 25mm respectively. Five days of treatment with Unani eye drops healed the corneal ulcer. The extent of this effect was comparable to that of moxifloxacin and was significant (p<0.05) when compared with that of the control group (Table 1).

Table 1. In-vitro antimicrobial effect of eye drop in corneal ulcer in rabbit’s eye

Group

(n=6)

Ocular lesions score (Mean ± SE)

Day 1

Day 2

Day 3

Day 4

Day 5

Control

8.3 ± 0.33

6.8 ± 0.31

6.2 ± 0.31

6.2 ± 0.31

4.3 ± 0.21

Eye drop

8.0 ± 0.37

6.8 ± 0.31*

5.3 ± 0.21*

4.3 ± 0.21*

2.3 ± 0.2*

Standard

8.0 ± 0.37

5.5 ± 0.22*

4.3 ± 0.21*

3.3 ± 0.21*

2.3 ± 0.21*

* p<0.05 as compared to the control group.

Discussion

Corneal ulcers are one of the commonest ophthalmic problems. Although promising chemotherapeutic agents have recently been developed, treatment of corneal ulcers constitutes a major therapeutic problem. Due to the injudicious and extensive use of antibiotics in modern therapy, antibiotic resistance has become common, including with respect to ocular pathogens.21 The antibacterial activity of Unani eye drop tested in-vitro using disc diffusion method and screened against S. aureus bacterial strain showed promising results. Dare hald, a chief ingredient of this eye drop formulation has been shown to possess antibacterial property22,23 against gram-positive and gram-negative bacterial species. The flowers of Azadirachta indica, another ingredient of formulation, also have antibacterial properties.24 Berberine, the chief alkaloid of Berberis aristata and Coptis teeta, possesses antibacterial and anti-inflammatory activities.25,26,27 A study conducted by Shahid28 found that Berberis aristata showed wide antibacterial activity against gram +ve bacteria. Another important imidazole alkaloid, chaksine has been isolated from Cassia absus Linn. shown to possess antibacterial activity.29 Coptis teeta Wall. possesses anti-inflammatory,30 antiseptic properties and also has antibacterial activity against common pathogenic bacteria.31 Rosa damascena Mill. is a valuable natural drug agent possessing bacteriostatic property.32 Rose water is used in conjunctivitis33 and has shown analgesic and antiseptic property which  may be of benefit in ocular diseases.34 Rose water obtained from petals of Rosa damascena Mill. is known for its soothing effect, and is also found to be beneficial in ophthalmopathy and has antibacterial property.35

Conclusion

All the above findings support the usefulness of the tested Unani eye drop formulation in the treatment of bacterial corneal ulcer such that its antimicrobial effect was comparable to that of moxifloxacin ophthalmic eye drops. The test drug seems to play an important role in the healing of corneal ulcer, most likely due to its anti-inflammatory and antimicrobial properties.

Acknowledgement

We are thankful to Ministry of Health & Family Welfare, Government of India for providing necessary facilities during this research work.

References

  1. Kafaru E. Immense help formative workshop. In Essential Pharmacology; 1st ed. Lagos, Nigeria: Elizabeth Kafaru Publishers; 1994.
  2. Lewis K, Ausubel FM.  Prospects of plant derived antibacterial. Nat Biotechnol 2006; 24: 1504-1507.
  3. Knauf HP, Silvany R and Southern PM. Susceptibility of cornea and conjunctival pathogens to ciprofloxacin. Cornea 1996; 15: 66-71.
  4. Ibn Sina. Al-Qanoon (Urdu translation by Ghulam Husain Kantoori), Matba  Munshi Naval Kishore Publication, Lucknow, India, 1887; vol. II, p. 187.
  5. Singhal GD and Sharma KR. Ophthalmic and otorhinological consideration in Ancient Indian Surgery. Singhal Publications, Allahabad, India, 1976; p. 21.
  6. Kadans JM. Encyclopedia of Medicinal herbs. Thorsons Publishers Limited, Wallingborough, Northamptonshire, 1970; p. 9.
  7. Biswas NR, Gupta SK, Das GK, Kumar N, Mongre PK, Halder D and Beri S.  Evaluation of Ophthacare Eye drops a Herbal Formulation in the Management of Various Ophthalmic Disorders. Phytotherapy Res 2001; 15: 618-620.
  8. Bonjar S. Evaluation of Antibacterial Properties of Some Medicinal Plants Used in Iran. J Ethnopharmacol 2004; 94: 301-305.
  9. Santos PRV, Oliveira ACX and Tomassini TCB. Controle Microbiogico De Productous Fitoterapicos. Rev Farm Bioquim 1995; 31: 35-38.
  10. Paul A K, Madan S and Gupta V. Clinical evaluation of an indigenous herbal eye drops preparation (Part I). Ind J Clin Prac 1992; 11: 58-60.
  11. Latif A. Amraz-e-ain “Humratul-ain”: A book of differential diagnosis of Red-eye, Publishers Fakhruddin Memorial Committee, Uttar Pradesh, 1985; p. 8-9.
  12. Bashar S, Hassan A and Omar S. “Tradition and Perspectives of Arab Herbal Medicine: A Review”, Evidence-based Complement Alternative Med 2005; 2: 475-479.
  13. Tayyab M. Ilmul-Ain aur Atibba-e-Qadeem, Muslim Educational Press, A.M.U., Aligarh, U.P. 1957; p. 2-5, 14, 40-42.
  14. Nadkarni KM. Indian Materia Medica, Popular Prakashan, Bombay, India, 2000; vol. I, p. 1072-1073.
  15. Dymock W, Warden CJH and Hooper D. Pharmacographia Indica, Trubner & Co. Ltd., 1891; vol. 2, p.257.
  16. Chopra RN, Chopra IC, Honda KL and Kapur LD. Indigenous drugs of India, Bankim Chatterjee Street,  Calcutta, IInd Ed., 1958; p. 292-293.
  17. Ghulam J. Kitab-ul-Murakkabat Almaroof Makhzan-ul-Muurakkabat. Ejaz Publishing House, New Delhi, India, 1995: 297.
  1. Baur AW and Kirby M.  Antibiotic Susceptibility Testing by Standardized Single Disc Method. Am J Clin Pathol 1996; 45: 493-496.
  2. Khan Attaullah.  Evaluation of Honey in the treatment of external bacterial infections of the eye. An Animals model. M.S. Thesis, Institute of Ophthalmology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, 1991: 31.
  3. Hogan M J, Kimura S J and Thygeson P. Signs and symptoms of uveitis. Anterior uveitis. Amer J Ophthal 1959; 47: 155-170.
  4. Ahmad S. Efficacy of Gentamicin and Neomycin on conjunctival bacterial flora. J Ophthal 1989; 8: 193-196.
  5. Mishal H. and Somani R. Herbal anti-bacterials, Hamdard Medicus, 2000; vol. XLIII: 109-112.
  6. Khan KH and Jain SK. Medicinal Plants -A retrospective, Hamdard Medicus, 2003; vol. XLVI: 23-33.
  7. Ross IA. Medicinal Plants of the World, Humana Press, Totowa, New Jersey. 2001; vol. 2, 81-95.
  8. Mekawi M. Effect of berberine alkaloid on cholera vibrio and its endotoxin. J Egypt Med Assoc 1966; 49: 554.
  9. Amin AH, Subbaiah TV and Abbasi KM. Berberine sulfate: Antimicrobial activity, bioassay and mode of action. Can J Microbial 1969; 15: 1067-1076.
  10. WOI. The Wealth of India ‘Raw Materials’ C.S.I.R., New Delhi, 2000; vol. 1: 135.
  11. Shahid M, Rahim T, Shahzad A, Tajuddin, Latif A, Fatima T, Rashid M, Raza A and Mustafa. Ethnobotanical studies on Berberis aristata DC. root extracts. African J Biotech 2009; 8(4): 556-563.
  12. Gupta KC and Chopra IC. A short note on anti-bacterial properties of chaksine: an alkaloid from Cassia absus Linn. Indian J Med Res 1953; 41: 459-60.
  13. Kareem N. Makhzanul Advia, Matba Munshi Naval Kishore, Kanpur, 1880; vol. 2: 347-348.
  14. WOI. The Wealth of India ‘Raw Materials’ C.S.I.R., New Delhi, 2001; vol. 2: 174-175.
  15. Basim E and Basim H. Antibacterial Activity of Rosa damascena Essential Oil. Fitoterapia 2003; 74: 394-396.
  16. Ghani HMN. Khazeenat-ul-Advia, Matba Munshi Naval Kishore, Lucknow, 1921; vol. 3: 500-501.
  17. The Unani Pharmacopoeia of India. Department of Ayush, Ministry of Health & family Welfare, Govt. of India, 2007; vol. 3: 31-33.
  18. Jafari M, Zarban A, Pham S and Wang T. Rosa damascena Decreased   Mortality in adult Drosophila. J Med Food 2008; 11: 9-13.