Quality Control of Certain Herbal Preparations Used for Respiratory Disorders in The Egyptian Market

Herbal Preparations

Herbal preparations used in this study were collected from different batches in the Egyptian market. The compositions of each herbal preparation are represented in Table 1.

Authentic Reference Materials

Pesticide standards: α-HCH, β-HCH, δ-HCH, Heptachlor, Heptachlor-epoxide, Aldrin, γ-Chlordane, Dieldrin, p,p'- DDE, Endrin, o,p'-DDT, p,p'-DDD and p,p'-DDT were purchased from Chem. Service, Inc (West chester, PA), supplied by Agricultural Pesticide Committee (APC), Ministry of Agriculture, Dokki, Giza, Egypt. Aflatoxins: Aflatoxins B1, B2, G1 and G2 were supplied from Mycotoxins Central Lab. and Food Safety, National Research Center, Dokki, Giza, Egypt. Heavy metals and microelements: Metals stock standards of Cd, Cu, Fe, Pb and Zn were obtained from Merck, Darmstadt, Germany (Merck's ampoules; 1000mg).

Media for Microbiological Study

Nutrient agar, MacConkey agar and Sabouraud dextrose. All of them are reconstituted and sterilized.

Micro-organisms

Bacterial strains: Bacillus cereus, B. polymexa, B. sphaericus, B. subtilis, Micrococcus spp. and Staphylococcus epidermidis. Fungi: Aspergillus candidus, A. niger, A. versicolor, Fusarium equiseti, F. oxysporum, Mucor pusillus and Penicillum spp were all supplied by Agricultural Pesticide Committee (APC), Ministry of Agriculture, Dokki, Giza, Egypt for microbial count measurements.

Sample Preparation and Instrumentation

Determination of pesticide residues: Plant extract prepared by immersion of 2g of the dry samples in 100ml of recently boiled distilled water for 5 minutes. Each sample was analyzed by Gas chromatography (GC) Hewlett Packard (HP) serial 6890, Ramsey, Minnesota, USA. Gas Chromatography equipped with different detectors, i.e. electron capture (ECD). Analysis of the pesticides was performed on two capillary columns, HP-5 (5%-phenylmethylpolysiloxane) and DB-35 (35%- phenylmethylpolysiloxane). Nitrogen was used as a carrier gas at a flow rate of 1ml/min. The temperatures of injector and interface were 250°C and 300°C, respectively. The temperature program for GC was as follow; initial temperature was 100°C for 1 min, raised at rate of 25°C/min to 170°C, isothermal for 1 min, raised at a rate of 3°C to 230°C, then isothermal for 1 min, finally raised at a rate of 8°C, then isothermal for 5 min. The Codex quality assurance criteria were followed to determine the performance of the multi-residue method. Recoveries and limit of quantitation (LOQ) were determined on samples at spiking levels. The average recoveries ranged between 81% and 104% and quantitation limits between 0.003 and 0.043 mg/kg [11, 12]. Determination of certain microelements and heavy metals: Samples have different batch numbers of Formula 1 and 2 are collected from different pharmacies for analysis of heavy metals, using atomic absorption spectrophotometric method [12, 13]. Using wet digestion method, 1.5g of each powdered sample were digested in Kjeldahl flasks set at 100 °c till complete digestion then diluted by deionized water and transferred quantitatively to 50ml volumetric flask. Filtrate was analyzed by Thermo Elemental model: Solar M Atomic Absorption Spectrophotometer was used for all the measurements, the current; wavelength and slit bandwidth of each element was adjusted automatically by the instrument software. Determination of microbial contaminants: Eighteen samples from different batches of herbal preparations (Formula 1 and 2) were examined for determination of microbial contaminants using viable count method. Viable count method One g of each of the herbal preparations was mixed separately, with 9 ml sterile peptone water and then 10- fold serial dilutions were made. Five level-spacing 1 logarithmic unit were investigated by pipetting 1 ml from each level in a plate, 15 ml of nutrient agar (agar for bacterial count, Sabouraud dextrose agar for fungal count and MacConkey agar for pathogenic coliform count), were added. The contents were allowed to solidify and inverted plates were incubated at 37ºC, examined after 2 days for both bacterial and coliform count, while for fungal count the plates were incubated at 28ºC, examined after 7 days. Suitable dilutions were counted [14]. Determination of aflatoxins: Determination of aflatoxins was carried out on herbal preparations (Formula 1 and 2) by blending 25g sample with 5g sodium chloride and 100mL methanol: water (80:20) using a high-speed blender jar for one minute, then filtered through fluted filter paper. 10mL of the filtrate were diluted with 40mL d water, and then filtered through a glass microfiber glass syringe barrel. Pass 8ml. and subjected to HPLC analysis. Waters Binary Pump Model 1525, a Model Waters 1500 Rheodyne manual injector, a Waters 2475 Multi-Wavelength Fluorescence Detector, and a data workstation with software Breeze 2 (USA). Filtered diluted to extract (0.8gm sample equivalent) completely through an Afla Test ®-P affinity column at a rate of 1-2 drops/second. Pass 10ml d water through the column at a rate of 2drops/second, elute the affinity column by passing one mL HPLC grade methanol through the column at a rate of 1-2 drops/second, collected in a glass vial, evaporated until dryness under a stream of nitrogen (immuno-affinity chromatography) [15].

Sample was derivatized using 100 µL of trifluoracetic acid (TFA), left for 15min. 900 µL of water: acetonitrile (9:1 v/v) were added and mixed well by vortex for 30 s, then the mixture was used for HPLC analysis using reversed phase column [Phenomenex C18 (250 x 4.6mm i.d.), 5 µm from water corporation (USA)]. An isocratic system with water: methanol: acetonitrile (6:3:1). The separation was performed at an ambient temperature at a flow rate of 1.0 mL/min. The injection volume was 20 µL for both standard solutions and sample extracts. The fluorescence detector was operated at wavelength of 360 nm for excision and 440 nm for emission [15].

GC/MS for analysis of volatile oils of herbal preparations: The essential oils were separately prepared by hydro distillation of 100 gm of dried powder from herbal preparations (Formula 1 and 2) and 400ml of each syrup (Formula 3 and 4) separately for four hours. The distillate in each was dried over anhydrous sodium sulphate and kept in the refrigerator until analysis. GC/MS analysis of the essential oil was carried out on Shimadzu Model GC-17A gas chromatograph interfaced with a Shimadzu model QP-5000 mass spectrometric detector. The instrument was controlled by the Shimadzu Class-5000 Version 2.2 software containing a NIST62 (National Institute of Standards and Technology) MS library. Volatiles were separated on a DB5-MS column (30 m length, 0.25 mm inner diameter, and 0.25 μm film thickness J&W Scientific, Santa Clara, Calif.). Injections were made in the split mode (54:1), the gas chromatograph was operated under the following conditions: injector 240ºC column oven 40ºC for 3 min, then programmed at a rate of 12ºC/min to 180 ºC, kept at 180 ºC for 5 min, and finally ramped at a rate of 40 ºC/min to 240ºC and kept for 5 min. Helium carrier gas, flow rate was 0.9 mL/min. The transfer line and ion– source temperatures were adjusted at 230 and 180 ºC, respectively. The HP quadrupole mass spectrometer was operated in the electron ionization mode at 70 eV. The scan range was set at 40-500 m/z. Peaks were first de- convoluted using AMDIS software (www.amdis.net) and identified by its retention indices (RI) relative to n- alkanes (C6-C20), mass spectrum matching to NIST library database and with authentic standards when available. The volatile components were identified by comparing their relative retention times and mass fragmentation patterns with those of the database libraries (Wiley7n.1 and wiley 7 NIST05 database) as well as the published data. The percentage of each component was determined by computerized peak area measurements. A series of authentic n-alkanes was subjected to GC under the same conditions, the retention time of each n-alkane was observed and Kovats index of each constituent of the oil was calculated by using special computer program. Norway).

Detection of Pesticide Residues

Persistent organic pollutants (POPs) include organic chemicals, such as the synthetic aromatic chlorinated hydrocarbons, which are only slightly soluble in water and are persistent or stable in the presence of sunlight, moisture, air and heat. In the past, they were extensively used in agriculture as pesticides. Thirteen pesticides were studied for identification and quantification. The detected residues organochlorine pesticides included: alpha-HCH, beta-HCH, delta- HCH, heptachlor, heptachlor-epoxide, aldrin, Gama chlordane, dieldrin, p, p-DDE, endrin, o.p- DDT, p, p-DDD and p, p-DDT.

present in (Formula 1) in concentration of 0.0025, 0.001 and 0.0002 mg/kg respectively. However, (Formula 2) contained only Heptachlor and p.p-DDT in concentration of 0.003 and 0.002 mg/kg. These results are incompliance with the international standards limit as the concentration of Heptachlor, Dieldrin and p.p-DDT should not exceed 0.05, 0.05 and 0.2 respectively [12, 16, 17].

By analysis of GC chromatogram (Figures 1a & 1b), it was found that Heptachlor, Dieldrin and p.p-DDT were

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Determination of Certain Microelements and Heavy Metals

Contamination of herbal materials with toxic substances can be attributed to many causes. These include environmental pollution (i.e. contaminated emissions from factories and leaded petrol and contaminated water, including runoff water, which finds its way into rivers, lakes and the sea, and some pesticides), soil composition and fertilizers. This contamination of the herbal material leads to contamination of the products during various stages of the manufacturing process [16, 17].

Samples have different batch numbers of Formula 1 and 2 are collected from different pharmacies for analysis of heavy metals. Results are tabulated in Table 2. It was found that all preparations contained Copper and Zinc. However, Cadmium and Lead are absent. Comparing the daily intake dose with the acceptable daily levels, it was concluded that the levels of Copper and Zinc did not exceed the maximum acceptable limits [18].

Determination of Microbial Contaminants

The need for the microbial quality control of commercial herbs is well clarified and recognized. This study aimed to evaluate the commercial herbs, as for their microbial contents. In addition, this study reflected the urgent need to reassess our methods of controlling the microbial contamination of commercial herbs. Total counts were used to assess sanitary quality, organoleptic acceptability, safety and utility of various herbal products. High viable count indicates poor sanitary quality, while low total counts do not necessarily carry the opposite implication [19].

Nine samples from three different batches of Formula 1 and 2 under study were represented by (A1, A2, A3, B1, B2, B3, C1, C2 and C3) and (A1o, A2o, A3o, B1o, B2o, B3o, C1o, C2 o and C3 o) respectively. The results obtained for bacterial and fungal count of both commercial samples were presented in Table 3.

The microbiological examination included the determination of total aerobic bacterial, total fungal and coliform counts as well as qualitative tests for identification of bacteria and fungi. Regarding Formula1 samples, it was found that the bacterial count ranged from 0–1x103 cfu/gm. Three samples out of nine were free from bacteria while fungal count ranged from 0 – 2x102 cfu/gm and six samples were free from fungi. Qualitative tests for bacteria showed that three samples had S. epidemidis, while the main fungal isolates were M pusillus, F. oxysporum and A. candidus along with presence of yeast.

Determination of Aflatoxins

Aflatoxins are a naturally occurring toxic metabolite produced by certain fungi (Aspergillus flavis and A. Parasiticus) However, A. flavis is common and widespread. These mycotoxins could produce hepatocarcinoma as a result of aflatoxicosis when given in very low doses for laboratory animals. Consequently, the exposure of humans of these aflatoxins has dangerous adverse reactions [20]. Conditions like increasing moisture content; high temperature and insect damage Tests for aflatoxins are designed to detect the possible presence of aflatoxins B1, B2, G1 and G2, which are highly toxic contaminants in any material of plant origin. HPLC chromatograms of aflatoxins in Formula 1 and 2 were presented in Figures 2a and 2b. It was concluded that the total concentration of aflatoxins in herbal preparations (Formula 1 and 2) is (0.52 μg/kg) and (1.63 μg/kg) respectively which is much lower than the acceptable limits.

of natural products are major factors for mould infestation and toxin production.

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GC/MS Analysis of Essential Oils of Different Herbal Preparations

Essential oils of four herbal preparations (Formula 1, 2, 3 and 4) were subjected to GC/MS analysis. Physical characters, yield %, total number of identified compounds and % of identified compounds in each herbal preparation was presented in Table 4 (Figure 3).

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Concerning Formula 3, seven main components were identified representing (97.34%) of the total detected compounds, the major components were thymol (47.84%) and anethole (34.73%) followed by linalool (6.25%) and α-terpineol (4.38%). All the compounds representing oxygenated monoterpenes. Twenty-four compounds were identified representing (81.9%) of the total detected compounds in formula 4. Thymol (63.24%) was the major component followed by anethole (9.33%) with monoterpenes and oxygenated monoterpenes representing (0.12%) and (78.76%) respectively. Identified components in the essential oil of each herbal preparation with their Kovat index and relative abundance are presented in Table 5.