INVESTIGATION OF STABILITY OF KOREAN GINSENG IN HERBAL DRUG PRODUCT Latifa

This study was designed to investigate long term st ability of K-Gin capsules, a herbal medication that contains korean ginseng. Stability studies ensuring the maintenance of product quality, safety and eff icacy throughout the shelf life are considered as pre-req uisite for the acceptance and approval of any pharmaceutical product. Conducting long term stabil ity studies following the guidelines issued by ICH, WHO and USP. The drug was stored under different cl imatic conditions: (25 ±1°C, humidity: 75%) Chamber 1(CH1); (30 ±1°C, humidity: 75%) Chamber 2(CH2) and (40 ±1°C, humidity 75%) Chamber 3(CH3) for 24 months,evaluation of capsulestchemica ly (ginsenosides Rg1 and Rb1), physicaly and microbiologicaly, besides determination of certain elements including heavy metals, detection of pesti cides residues and mycotoxins. The activation energies we re found to be 10.99 and 10.2 (k.cal.mol ) for Rg1 and Rb1 respectively. The calculated t 1/2 values for Rg1 were found to be 94 months in (CH1), 68.4 months in (CH2) and 51 months in (CH3). For Rb1 were found to be 100.3 months in (CH1), 73.39 months in (CH2) and 56.7 months in (CH3). Capsules assay results fo r Rg1 after 24 months period of study were found to be 98.93% in (CH1), 97.13% in (CH2) and 96.43% in (CH3 ). For Rb1 96.93% in (CH1), 96.83% in (CH2) and 96.13% in (CH3). Studies on the elemental compositi on confirmed absence of toxic heavy metals. Microbiological studies revealed no significant cha nges in total viable aerobic count of bacteria and fungi. The capsules were found to be free from any pestici des residues and the mycotoxins content was 723 ng kg. The methods involved in this study covered, High Performance Liquid Chromatography (HPLC), Atomic Absorption Spectroscopy (AAS), Gas Chromatog raphy-Mass Spectrometry (GC-MS) and ELISA. The results were found to be within the allowable l imits stated in the guidelines. From this study K-G in capsules were proved to be stable chemicaly and phy sical under studied storage conditions for 24 mont hs.


INTRODUCTION
Ginseng is a well-known medicinal herb in traditional Asian medicine and is considered an adaptogen. Panax ginseng C.A. Meyer (Araliaceae), grows in China and Korea and more than 30 ginseng saponins (ginsenosides) have been isolated as the main biologically active constituents that include anti-carcinogenic, anti-diabetic and anti-inflammatory effects, as well as cardiovascular protection and neuroprotection (Zhang et al., 1996;Yun et al., 2001;Joo et al., 2005;Jung et al., 2005).
As cancer is quickly becoming the leading cause of death in the world, many reports have focused on chemoprevention trials with Panax ginseng. Most of the pharmacological actions of ginseng are attributed to a variety of ginsenosides, which are triterpenoid saponins (Attele et al., 1999;Huang et al., 2005). The physiological and medicinal effects of the various Science Publications AJAS ginsenosides differ and can even be oppositional (Sengupta et al., 2004;Joo et al., 2005). Since ginsenosides produce effects that differ from one another and a single ginsenoside initiates multiple actions, the overall pharmacology of ginseng is complex. Thus, ginseng extracts have been studied to examine the final activity of a wide range of biological actions (Tsang et al., 1985;Nishino et al., 2001;McElhaney et al., 2004).
Korean Ginseng contains adaptogens that have been shown to return the body's system levels back to normal by equalizing body's system levels and has been used for the following: To lower cholesterol, increase metabolism, increase energy, stimulate the immune system, alleviate fatigue, reduce nervousness and reduce stress on the body (Bucci, 2000).
Korean ginseng is officially registered in Saudi Arabia for its anti-diabetic and anti-inflammatory effects, as well as cardiovascular protection with a recommended dose of 700 mg twice daily. Stability data is not reported in the official registration files of Korean ginseng. Therefore, the present study was designed to investigate the stability of Korean ginseng capsules, besides carrying the general chemical, physical and microbiological quality control measurements for it.

Standard Solution
A solution each of 0.5 mg mL −1 of ginsenoside Rg1 and ginsenoside Rb1 reference materials in methanol were prepared.

Preparation of Test Sample
1 gm of the powered drug was weighed extracted with 70 mL of 50% (v/v) solution of methanol, boiled in water bath under reflux for one hour. Then cooled centrifuged, evaporated to dryness under reduced pressure at a temperature not exceeding 60ºC. The residue was dissolved in 10 mL buffer solution containing 3.5 gm of sodium dihydrogen phosphate in 1000 mL of water (solution A). Five ml of solution A was applied to the cartridge (clean up procedure using solid phase extraction containing 360 mg of octadecylsilyl silica gel for chromatography), eluted with 20 mL of water, followed by 15 mL of 30% (v/v) methanol. The elute was discarded and then the cartridge was eluted with 20 mL of methanol. The methanol extract was evaporated to dryness. The residue was redissolved in 2 mL of methanol before injection.

Elemental Analysis
Atomic absorption spectroscopy was performed using Shimadzu AA-6800 (AAS) with graphite and flame auto switch over mode, ASC 6100 auto sampler and HVG1 hydride generator was used. The amount of each element was determined by using appropriate lamp. The content of the respective element was calculated by using a calibration graph. Range of wavelengths: 190 to 900 nm.

Preparation of the Working Standard Solution
Standard solutions (1 mg mL −1 ) of Pb, Hg ,Na, Ca, Cr, Cd, As, K, Mg, Cu, Mn, Fe obtained from Sigma-

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Aldrich Chemie GmbH, Germany were used to prepare the standard solutions. Standard solutions were diluted, with 0.1M HNO 3 to a range of standards that covers the linear range of the element to be determined.

Preparation of Test Sample
One g of the sample was weighed, dried, ached. The residue was dissolved in 30 mL of 0.1M HNO 3 . Blank was treated in the same way as product.

Effect of Strong Acids, Alkalies and Temperature
The effect of strong acids, alkalies and temperature were studied by treating dilute samples solutions of ginsenosides Rg1 and Rb1 (0.5 mg mL −1 each) with both 1 M sodium hydroxide and 1 M HCl. The experiments were conducted at 60±1°C for 1 h, Samples were analysed by the previously described HPLC method.

Effect of Phosphate Buffer pH 7.4
The catalytic effect of phosphate buffer pH 7.4 (plasma pH) at 60±1°C for one hour was studied. The same concentration of ginsenosides Rg1 and Rb1v (0.5 mg mL −1 each) was used for the study. Samples were analysed by the previously described HPLC method.

Disintegration Time of Capsules
The test was carraied according to USP Disintegration test.

Extraction and Clean up Procedures
Various extraction and clean up procedures have been used in the detection of pesticides as described by (Yoon et al., 1999;Khan et al., 2001).

Recovery Experiment
The samples were extracted with hexane. The extracts were cleaned up by Solid Phase Extraction (SPE), using Florasil columns and hexane and hexan/CH2Cl2 (17: 3) as eluents. The standard was processed following the same procedure (Yoon et al., 1999;Khan et al., 2001). The eluates were analyzed by GC-MS under the following conditions: Column: (1

Microbiological Studies
The drug sample was tested using the standard protocol for the total viable aerobic count of bacteria and fungi to check the possible presence of Escherichia coli; Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella. (CE, 2004)

Mycotoxins Determination
K-Gin capsule was tested in duplicate for the aflatoxins content, which are naturally occurring mycotoxins. A quantitative evaluation of aflatoxin were carried out through antigen antibody reaction using Enzyme Linked Immunosorbent Assay (ELISA) technique on minilyser (fully automated system), modified Tecan RSP 5051 by Tecan company using RIDASCREEN aflatoxin total screening kit (Art. No. 470) manufactured by R-Blopharm AG, Darmstadt, Germany.

Test Procedure
The tested samples were finely ground and shaken with 70% (v/v) methanol and filtered. The aflatoxin standards, samples dilutions, enzyme labeled aflatoxin (enzyme conjugate) and anti-aflatoxin antibodies were added in the wells in the microtiter strips which were coated with specific antibodies to mouse antibodies. Free and enzyme labeled aflatoxin competed for the antibody binding sites of the anti-aflatoxin antibodies, which themselves were bound simultaneously by the capture antibodies on the microtiter plate. Any unbound enzyme conjugate was removed in a washing step. Enzyme substrate (urea perroxide) and chromogen (tetramethylbenzidine) were added to the wells and incubated. Bound enzyme conjugate converted the colorless chromogen into a blue product.
By the addition of the stop reagent, the color changed from blue to yellow. The measurement was made photometrically at 450 nm (optical reference wavelength ≥600 nm). The absorption is inversely proportional to the aflatoxin concentration in the sample.

Elemental Analysis
K-Gin capsules were found to contain sodium(2718 µg g −1 ), magnesium(235 µg g −1 ) and relatively large amount of potassium(15095 µg g −1 )and it is rich in calcium (30831 µg g −1 ). No other s specially heavy metals were found.

Effect of Temperatures
The effect of temperatures were presented in the following tables and figures

Effect of Alkalies, Acids and Phosphate
Buffer pH7.4 on Stability of Ginsenosides Rg1 and Rb1

Physical Examination
The K-Gin powder and shell were examined over the period of 24 months. The powder retained its brown color and no distinctive odor was noticed except in CH2 and CH3 after 24 months where the color changed to dark brown. The capsule shell remained transparent and colorless over the same period except for samples in CH2, CH3 condition after 24 months the capsules powder was obsersved change to hard cylindrical lumps.
Disintegration time was found to be between 5 and 6 min.

Microbiological Studies
The results of microbiological studies for K-Gin revealed no significant changes in total viable aerobic count of bacteria and fungi. The total count of viable aerobic bacteria and fungi was found to be less than 10 colony forming unit/gm (fcu) after 24 month period of study, regardless of storage conditions. The results also indicate that the primary pathogens Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella were not present in the tested samples of K-Gin capsules.

Aflatoxin Results
The mean concentration of total aflatoxin contents in K-Gin capsules was found to be 723 ng kg −1 .

Introduction
It is ageneral belive even, a mong health profisionals that herbal product formulations are safe used without limitation. People this believe that takes these formulations are among the over counter preprations that can be sold freely; however for quality control complete assessment of such preprations should be investigated covering physical, chemical, microbiological, toxicological and theraputic stability to insure evedencebased safety (Thakur et al., 2011). Accordingly this study was carried to one type of herbal product.
The quality control of herbal products forms one of the difficulties for the analysts due to their complex matrixs. Methods used for the assays of active constituents in these preprations should be precise,

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AJAS accurate and stability indicating, therefor proper validation of these methods should be carried. HPLC forms one of the best methods that can be applied for such assay. Any results obtained should be properly evaluated to insure safe, non toxic herbal product.

Method Validation
Under the chromatographic conditions described, ginsenosides Rg1 and Rb1 peaks were completely resolved Fig. 1 and 2. Calibration curves constructed from the peak areas and the corresponding ginsenosides concentrations, in each calibration standard were linear from 0.1 to 0.7 mg mL −1 . The mean slope and intercept for the different calibration curves of gisenosides Rg1 and Rb1 are presented in Fig. 3 and 4. The correlation coefficients were 0.9986 and 0.9967 respectively. The RSD% were found to be 0.0014 and 0.574%, limit of detection and limit of quantitation were e 7.5 and 30 µg mL −1 respectively.
Method validation establishes that the method performance characteristics are suitable for the intended use, as shown in Table 1.

Elemental Analysis
K-Gin capsules were found to contain sodium, magnesium and relatively large amount of potassium and it is rich in calcium, this is in agreement with the finding reported by (Jackson et al., 2004). No other elements especially heavy metals were found. The herb therefore is considered a suitable supplement for K and Ca.

Effect of Temperature
From the results obtained it is noticed that ginsenoside Rg1 and ginsenoside Rb1 in K-Gin capsules are stable over the range of 25, 30 and 40ºC with humidity 75%. Ginsenoside Rg1 and ginsenoside Rb1 in K-Gin capsules undergoes slight decomposition and lose about 0.17, 0.47 and 1.17% for ginsenoside Rg1 and 0.17, 0.67 and 1.47% for ginsenoside Rb1 after six months at CH1, CH2 and CH3 respectively. The rate of decomposition increases more and it reaches around 1.07, 2.87 and 3.57% for ginsenoside Rg1; and 3.07, 3.17 and 3.87% for ginsenoside Rb1 after 24 months storage in CH1, CH2 and CH3 respectively Table 2.
However, it is worth noting that the ginsenoside Rg1 and ginsenoside Rb1 standards were unstable as raw materials over the range of 25, 30 and 40ºC with humidity 75%. Ginsenoside Rg1 and ginsenoside Rb1, both, undergo slight decomposition under these conditions, ginsenoside Rg1 loses about 4.67, 6.1 and 6.98% and ginsenoside Rb1 loses about 3.63%, 6.45% and 9.96% after six months storage in CH1, CH2 and CH3. The rate of decomposition increases with time. The loses reached around 15.8 21.6 and 26.5% for ginsenoside Rg1 and around 15.25, 20.51 and 26.25% for ginsenoside Rb1 respectively Table 3. The kinetic parameters for the degradation of ginsenoside Rg1 and ginsenoside Rb1 under different temperatures 25, 30 and 40ºC with humidity 75% were found to follow first-order reaction. The semi-logarithmic plot of log (a/a-x) against time (t) were found to give straight line with slopes of 0.0032, 0.0044 and 0.0059 under CH1, CH2 and CH3 conditions respectively for ginsenoside Rg1 as shown in Fig. 7 and for Rb1 0.003, 0.0041 and 0.0053 CH1, CH2 and CH3 respectively as shown in Fig. 8, using the formula for the first-order reaction kinetics kT a log 2.303 a x = − (Connors, et al., 1986).The reaction rate constant (K) was found to be 0.00737, 0.011 and 0.0136 month −1 for samples from CH1, CH2 and CH3 respectively for ginsenoside Rg1 and 0.0069, 0.0094 and 0.0122 month −1 for CH1, CH2 and CH3 respectively for ginsenoside Rb1. The half-life time t 1/2 was found to be 94, 68.4 and 51 months for CH1, CH2 and CH3 respectively for ginsenoside Rg1 and 100.3, 73.39 and 56.7 months for CH1, CH2 and CH3 respectively for ginsenoside Rb1 Table 4 and 5.
Agraph of the logarithm rate constant (K) against the reciprocal of temperature1/T is rectilinear with a slope of -Ea/2.303R and an intercept at 1/T = log A (Arrhenius plot) (Fig. 5 and 6), from which the activation energy was calculated and was found to be 10.99 kcal mole −1 and 10.2 kcal mole −1 for ginsenoside Rg1 and ginsenoside Rb1 respectively as shown in Table 4 and 5.

Effect of Alkalies, Acids and Phosphate Buffer pH7.4 on Stability of Ginsenosides Rg1 and Rb1
It is reported that acid hydrolysis of ginseng result in degradation of ginseng, causes epimerization, cyclization and hydration of 20s-protopanaxadiol (sppd) and 20s-protopanaxtriol (s-ppt) (Han et al., 1982). Fig. 10 confirms this result as compared to the control Fig. 9.
While ginseng was found unstable in acid, however it is stable in alkalies and phosphate buffer pH 7.4. The findings in this study is confirm with the results obtained by Yu et al. (2007).      Time  CH1  CH2  CH3  CH1  CH2  CH3  0 100.

Physical Examination
The capsules retained their appearance throughout the studies and the disintegration time was found between 5 and 6 min, which is suitable enough for quick contents absorption within short time to exert their action.

Pesticides Residues
K-Gin capsules were found to be free from any pesticide residues, this is in agreement with the finding reported by (Durgnat et al., 2005).

Microbiological Studies
The results demonstrate absence of microbiological contamination in K-Gin capsules, this is in agreement with the finding reported by (Jackson et al., 2004)

Aflatoxin Contents
The result of aflatoxin contents in k-Gin capsules was compared with the specification limits outlined in the Saudi Arabian Standards Organization No. 1151/1998 and G.C.C. Organization for Standardization and Methodology No. 841/1997 and USP limits which is ranging from 800-4300 ng kg −1 , NMT 20ppb, respectively and the result was within the allowable limits.
In a previous work we reported the freedom of the product from toxicity (Gadkariem et al., 2010).

CONCLUSION
Assessment of the stability, quality and safety of finished herbal drug products is an important and challenging issue for health professionals.
The results obtained revealed that the drug product included in the current study was found to be safe on several aspects. They were free from toxic heavy metals, pesticides residues and primary pathogens. It is worth mentioning that in all these drug product mycotoxins was lower than the allowable limits.
The stability results obtained during the current study supported the rule of two years shelf life for K-Gin registered in Saudi Arabia.

RECOMMENDATION
According to our findings, This type of rigorous investigations should be carried out for all herbal products sold in pharmacies or herbal shops. Future research in such preprations should include cilinical feedback to insure the safety of such preprations.
In cases where the manufacturers claim and apply for increase in the shelf life of their product (more than two years), they have to provide more data regarding the safety and stability of their product, covering the desired period of time, in the drug registration files. Because of the complex nature of the herbal drugs, their recommended storage conditions must be included and specified on the drug container.