In last few years exploring of herbs used for medicinal purposes, which may be safe, economical and easily available to the consumers as well as resistance of disease, causing organisms should be minimum or no development of resistance. The efficacy of more plant species has been determined through scientific validation but most herbal remedies used may cause serious toxic effects and also drug-drug interaction because these herbal remedies may not undergo careful scientific evaluation. Ongoing researches are required to explain the pharmacological activities of many herbal remedies being used [1]. As the environmental conditions, such as the type of soil and cultivation, methods may affect the natural composition of the constituents in a plant so the ingredient that may cause a specific antimicrobial effect is not known. The antimicrobial activity of most plants have been determined but majority of them have not been evaluated properly [2]. A source of antimicrobial activity has been reported from many plants therefore these plants may be used as a source of effective and powerful drugs in different countries of the world [3]. Ointments containing Aloe vera, Neem and Turmeric exhibited broad spectrum anti-bacterial and anti-fungal activitites against E. coli, S. aureus, Pseudomonas aeruginosa, B. subtilis. A. varis, A. niger and P. notatum [4]. Medicinal plant parts may be collected either in small quantities for use by the local communities and folk healers or in larger quantities for many herbal manufacturers for extracts as raw drugs because they possess different medicinal properties [5]. The main objectives of the current study were, to evaluate the antimicrobial branded herbal formulations for their efficacy and awareness of the public about blind use of these herbal formulations.

This research study was performed at the Department of Microbiology, Hazara University, Mansehra, Pakistan. All the experimental equipment and materials were supplied by the said department.

Samples collection

Forty six herbal branded formulations were purchased from the local market.

Apparatus used

Pre sterilized glass petri dishes, Metallic borer, volumetric flask, Pyrex A (Germany), Sanyo labo autoclave, MLS-3780, S.NO-2Y0301, Phase, Company Sanyo electric co, Ltd Made in Japan, Streamline Horizontal laminar flow cabinet, ESCO. En 1822.1 class H 13. HEPA filters, ISO 14644.1 Class 4. IEC 61010-1, US Federal standard 209E Class 10, Uni-Bloc SHIMADZU. Capacity Maximum 220 g and Minimum 10mg. SHIMADZU corporation made in Japan, Irmeco hybridization Oven. Digital constant temperature tank (China). Item- Model VRN-360. RPM 60-230 +10, Gemmy industrial corp (Taiwan).

In vitro antimicrobial studies of selected herbal branded formulations

The herbal branded formulations were tested by Agar Well Diffusion Techniques as described by [6]. Different concentrations of the extract were used for testing. For the agar well diffusion method, Mueller Hinton agar plates were seeded with standard inoculation of the microorganisms being tested. Herbal branded formulations were studied for their antimicrobial activity against the selected experimental microorganisms.

Applications of herbal medicines

Wells of 6 mm diameter were prepared in the plates with a sterile metal borer and five, 10, 15, 20, 25 and 100 microlitres of the already prepared test samples were pipette directly into the wells. These plates were incubated overnight at 37°C [7]. The antimicrobial potential of test compound was determined on the basis of diameter of zone of inhibition around the wells [8]. The test was carried out in triplicate and the mean zones of inhibition were calculated. For each bacterial strain, controls were included that comprised of sterile water (in the case of the aqueous extract) or ethanol (in the case of the ethanolic extract). Other wells were supplemented with 10 µg ciprofloxacin. Griseofulvin antifungal discs were used as standard drug. The zones of inhibition were then measured after 24 hrs incubation period. All the experiments were conducted in triplicate [9]. In case, ethanol was used as solvent, calculated the effective zone size of the test samples the zone size of the negative control (ethanol) was subtracted from the total zone of inhibition size.

Antibacterial effects against Escherichia coli

Twenty-eight herbal products were tested for their antibacterial activity against E. coli. All the tested products were clinically used against all pathogenic condition caused by E. coli. The manufacturer of these tested herbal formulations claim that these herb are used for various infections. It is clear that the drops of B14 has maximum inhibitory activity against this gram negative bacterium with zone of inhibition 25 mm. The syrup B8 showed weak activity against E. coli with mean zone of inhibition 7 mm. B11 and B41 also showed activity against the tested bacterium with zone of inhibition 15 and 13 mm respectively. The antibacterial effects of these products were compared with standard broad spectrum antibacterial drug (ciprofloxacin) having 26 mm zone size. It was very surprising to note that twenty four tested products failed to show antibacterial activity although the label of all these products indicated that these products were being used in various bacterial infections (Table 1).

Table 1: Antibacterial potential of various herbal branded formulations against Escherichia coli.

Antibacterial effects against Staphylococcus aureus

Twenty-seven herbal formulations were tested against S. aureus. Out of tested samples only B11 exhibited antibacterial effects against S. aureus with zones of inhibition 20 mm and rest of samples were devoid of antibacterial effect (Table 2).

Table 2: Antibacterial potentials of various herbal branded formulations against Staphylococcus aureus.

Antibacterial effects against Salmonella paratyphi

Five herbal products were tested against S.paratyphi as the labels of these products indicated that these products are recommended in the treatment of S. paratyphi infections. Out of tested products B34 and B30 exhibited significant effect against tested bacteria. The zone of inhibition of B34 and B30 are 19 and 17 mm, respectively (Table 3).

Table 3: Antibacterial potentials of various herbal branded formulations against S. paratyphi.

Antibacterial effects against Shigella dysenteriae

Fifteen herbal branded formulations were tested for their antibacterial effects against Shigella dysenteriae. Among the tested products only B11 showed significant activity had mean zones of inhibition 19 mm and rest of antimicrobial products showed resistance. The activity of the test samples were compared with Standard anti-bacterial drug with zone of inhibition 20 mm (Table 4).

Table 4: Antibacterial potentials of various herbal branded formulations against Shigella dysenteriae.

Antibacterial effect against Pseudomonas aeruginosa

Twenty-five herbal formulations were tested against P. aeruginosa. These formulations are indicated against P. aeruginosa infections. Among the tested samples only B18 was active with mean zone of inhibition 12 mm (Table 5).

Table 5: Antibacterial potential of various herbal branded formulations against Pseudomonas aeruginosa

Antibacterial effects against Salmonella typhimurium

None of the tested formulations were effective against Salmonella typhimurium (Table 6).

Table 6: Antibacterial potential of various herbal branded formulations against Salmonella typhimurium.

Antifungal activity against Candida albicans

Ten herbal branded formulations were tested for the determination of their antifungal activity. Out of the tested products four products showed activity against Candida albicans with zone of inhibition 26 mm, 5 mm, 4 mm and 6 mm respectively. Surprisingly B14 showed better result than Griseofulvin the standard anti-fungal drug (Table 7).

Table 7: Antifungal activity against Candida albican.

The scope of present study is to evaluate the antimicrobial activity of various herbs in the form of final dosage form. Herbal products are available in different dosage form like syrups, capsules, tablets, powder, ointments, emulsions and herbal teas. A number of herbal manufacturing companies have surfaced and established well equipped manufacturing units throughout Pakistan. Some of them are enjoying a very good business position while others are struggling. Botanical medicine has become a topic of increasing global importance, with both medicinal and economic implications. The numerous reports of adverse effects and widespread sale of adulterated products and misleading health claims of these products demand proper regulations on botanical medicine [10]. Depending on the particular country and existing legislation, herbal products used for diagnosis, cure, mitigation, treatment, or prevention of diseases are normally regulated as drugs. However, in some countries, including the United States, botanical products are marketed as "dietary supplement". Other countries treat the herbal preparations as drugs, and to be registered these products need to be tested to prove their safety and clinical efficacy. However, so far, few programs have been established to study the safety and efficacy of herbal medicines as originally proposed by the WHO Guidelines for the assessment of herbal medicines [11,12]. Currently, no organization or government body regulates the manufacture or certifies the labeling of herbal preparations. This means there is no assurance that the amount of the herb contained in the bottle or even from dose to dose, is the same as what is stated on the label. However, it is still important to ask companies that are making standardized herbal products the basis for their product's guarantee. It is still important to consult a doctor or an expert in herbal medicine for the recommended doses of any herbal products [13]. Drug regulatory agency (DRA) of Pakistan has recently been established and is in process of completing its structure. The tasks assigned to DRA were previously carried out by Ministry of Health of Pakistan (MOH), which has been dissolved. Affairs related to drugs/ medicines are governed by Drug Act of Pakistan 1976 with few amendments. But Drug Act of Pakistan 1976 only addresses the Allopathic medicines, while the Herbal/Unani/Homeopathic medicines do not qualify the definition of drug as per Drug Act. A drug may be any substance or mixture of substance that is manufactured, sold, stored, offered, for sale or represented for internal or external use in the treatment, mitigation, prevention or diagnosis of disease, an abnormal physical state or the symptoms thereof in human being or animals or the restoration, correction or modification of organic function in human being or animals. Not being a substance exclusively used or prepared for use in accordance with the Ayurvedic, Unani, Homeopathic or Biochemical system of treatment except those substances and in accordance with such condition as may be prescribed [14,15]. Tables 8-12 shows list of herbal products purchased from the market and their ingredients (herbs). Literature review in Table 1 shows all these herbs have a strong research data for their pharmacological activities. Almost every sample product contains one or more herbs having reported antimicrobial activity supporting the label claims of these products. But the present study shows that only few products have shown promising results against the selected strains.

Table 8: List of herbal formulations used for the treatment of GIT disorders. (Colitis due to dysentery, diarrhea, gastritis and enteritis, acute and chronic dysentery, useful for intestinal ulcers, Cholera. For watery and bloody diarrhea and gastrointestinal weakness and cramps, gastralgia, peptic ulcer).

Table 9: List of herbal formulations used for Respiratory Tract Infections ( RTIs ).

Table 10: List of herbal formulations used for Urinary Tract Infections (UTIs)

Table 11: Medicines used for treating different type of temperatures.

Table 12: Medicines used for different types of skin disease.

The results of the present study indicated the poor quality of herbal products being sold in the market with very high label claims. Only 9 out of 46 sampled products with antimicrobial label claimed qualify to some extent for medicinal use. As any health authority (DRA/MOH) in Pakistan does not regulate the herbal products, there was no control or check on the manufacturers and prescriber of these medicines. All the tested products contained herbs, which had proven data for their antimicrobial activity, still most of them failed to produce any pharmacological results. We can conclude any of the following reasons.

1. These products had not been produced by the professional people.

2. They had not been processed properly.

3. Selection of herbal ingredients was not proper. Quality assurance played important role to monitor such shortcomings.

4. Common extraction processes for herbal product manufacturing were decoction, distillation, infusion, maceration and percolation. These processes need trained employees and appropriate equipment. Any deficiency in provision of both of these may lead to poor quality.

It is necessary to identify and rectify these problems immediately. Following recommendations were made based on present study:

1. Proper legislation must be done to regulate the manufacturing and prescription of Herbal/ Unani/Homeopathic medicines.

2. Only qualified pharmacists must be allowed to supervise the manufacturing of such products. The role of Botanist/Phytochemist is necessary for proper identification of herbs and their extraction process.

3. The herbal manufacturing industries should be issued manufacturing licenses as is done in case of pharmaceutical industries.

a. Herbal manufacturing industries should be forced to establish well equipped quality control labs to ascertain the efficacy and safety of their products.

4. Quality assurance is required to monitor all the activities related to identification, collection, drying, transportation, storage, and extraction of herbs, manufacturing of herbal product, stability of formulation, quality control and marketing.

5. As the properties of soil to soil vary it is impossible to procure or collect herbs with similar phytoconstituents every time, every industry must have its own Research and Development department (R&D).