DRAFT 2: Major Project

            Hron et al. (1999) constructed a study to determine the amount of (+)-, (-)- and total gossypol in cottonseed using high performance liquid chromatography (HPLC) technique. Gossypol is the major active compound that causes temporary infertility in males. Gossypol in sufficient quantity can be toxic to animals. Bond connecting with the two naphthyl rings lead to steric hindrance between the functional groups of the molecule explaining the availability of both (+)- and (-)-isomers. Thus, scientists attempted to separate these two isomers. In Hron et al. (1999) study, acid-delinted cottonseed samples were selected to investigate in this study. The samples were ground and cracked, and then meats were separated out. The samples of meats were accurately weighed and derivatized by (R)-(-)-2-amino-1-propanol. A quantitative HPLC procedure was developed for the separation of (+)- and (-)-gossypol contained in cottonseed. An octadodecylsilane was used as reverse-stationary phase and eluted with acetonitrile: 20%potassium hydrogen phosphate (pH 3.0) (ratio 8:2) at 1.0 milliliter/minute. The (+)- and (-)-gossypol-2-amino-1-propanol complexes were separated at 1.4 and 2.6 minutes, respectively. The results showed that a crop of Upland cottonseed had (+)-enantiomer more than (-)-enantiomer, 67.6% and 32.4%, respectively; whereas, a seed sample from a Pima crop in Texas showed a higher amount of (-)-gossypol than (+)-gossypol, 53.1% and 46.9%, respectively. In conclusion, the HPLC method can distinguish gossypol from gossypol-like compound and quantify them while the official American oil chemist societies (AOCS) method can only detect total amount of gossypol. The researchers suggested that their new HPLC method can conveniently and quantitatively determine the isomers of gossypol in cottonseed. Moreover, they assumed that this HPLC method might possibly be scaled-up to provide materials for antiviral activity testing.

            This study provides scientific evidence to support herbal medicine in term of the quantitative analysis of each enantiomers of gossypol in medicinal plants which is necessary for herbal drug development. However, there are some limitations.

1)      The plant authentication should be carefully investigated. According to Mukherjee (2005), the specimen of plant should be compared with herbarium reference voucher specimens, or authenticated by a botanist to avoid mistakes over plant identification.

2)      This study had an objective to quantify the amount of active compound in medicinal plant. In experimental part, the researcher only described about the topic of linearity, accuracy and precision. ICH guideline (2005) summarized that the method validation was good practice for quantitative analysis. It was ensured that the analytical methodology was valid and reliable. Thus, the researchers should completely do in method validation for the most suitable method in quantifying the gossypol isomers.

3)      The researchers did not explain the method of AOCS in the part of experimental procedures. They did not use the statistical parameters to determine the differences of the result among the two methods; therefore, the researchers might have bias on data conclusion in explaining how this HPLC method was more suitable than AOCS method. They should have used pair t-test for determining the fitting of data.

The strength of this study is that HPLC technique is broadly accepted for many laboratories because HPLC has high reproducibility, high resolution, and can be detected in part per million levels. Moreover, another advantage of this research study is the clearly results that included text, tables and figures which were not required lengthy and complex explanation.

References

Hron, R., Kim, H., Calhoun, M., & Fisher, G. (1999). Determination of (+)-, (−)-, and Total Gossypol in Cottonseed by High-Performance Liquid Chromatography. Journal Of American Oil Chemist's Society, 76(11), 1351 - 1355.

Mukhejee, P.K. (2005). Quality control of herbal drug: an approach to evaluation of botanicals.
            New Delhi: Galaxy Printers.

International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Uses. (2005). Validation of Analytical Procedures: Text and Methodology Q2(R1). (pp. 1-13). Geneva: ICH.

DRAFT 1: Major Project

     Hron et al. (1999) constructed a study to determine the amount of (+)-, (-)- and total gossypol in cottonseed using high performance liquid chromatography (HPLC) technique. Gossypol is the major active compound that causes temporary infertility in males. Gossypol in sufficient quantity can be toxic to animals. Bond connecting with the two naphthyl rings lead to steric hindrance between the functional groups of the molecule explaining the availability of both (+)- and (-)-isomers. Thus, scientists attempted to separate these two isomers. In Hron et al. (1999) study, acid-delinted cottonseed samples were selected to investigate in this study. The samples were ground and cracked, and then meats were separated out. The samples of meats were accurately weighed and derivatized by (R)-(-)-2-amino-1-propanol. A quantitative HPLC procedure was developed for the separation of (+)- and (-)-gossypol contained in cottonseed. A octadodecylsilane was used as reverse-stationary phase and eluted with acetonitrile: 20%potassium hydrogen phosphate (pH 3.0) (ratio 8:2) at 1.0 milliliter/minute. The (+)- and (-)-gossypol-2-amino-1-propanol complexes were separated at 1.4 and 2.6 minutes, respectively. The results showed thata crop of Upland cottonseed had (+)-enantiomer more than (-)-enantiomer, 67.6% and 32.4%, respectively; whereas, a seed sample from a Pima crop in Texas showed a higher amount of (-)-gossypol than (+)-gossypol, 53.1% and 46.9%, respectively. In conclusion, the HPLC method can distinguish gossypol from gossypol-like compound and quantify them while the official American oil chemist societies (AOCS) method can only detect total amount of gossypol. The researchers suggested that their new HPLC method can conveniently and quantitatively determine the isomers of gossypol in cottonseed. Moreover, they assumed that this HPLC method might possibly be scaled-up to provide materials for antiviral activity testing.

            This study provides scientific evidence to support herbal medicine in term of the quantitative analysis of each enantiomers of gossypol in medicinal plants which is necessary for herbal drug development. However, there are some limitations.

1)      The plant authentication should be carefully investigated. According to Mukherjee (2005), the specimen of plant should be compared with herbariumreference voucher specimen, or authenticated by a botanist to avoid mistakes over plant identification.

2)      This study had an objective to quantify the amount of active compound in medicinal plant. In experimental part, the researcher only described about the topic of linearity, accuracy and precision. ICH guideline (2005) summarized that the method validation was good practice for quantitative analysis. It was ensured that the analytical methodology was valid and reliable. Thus, the researchers should completely do in method validation for the most suitable method in quantifying the gossypol isomers.

3)      The researchers did not explain the method of AOCS in the part of experimental procedures. They did not use the statistical parameters to determine the differences of the result among the two methods; therefore, the researchers might have bias on data conclusion in explaining how this HPLC method was more suitable than AOCS method. They should have used pair t-test for determining the fitting of data.

The strength of this study is that HPLC technique is broadly accepted for many laboratories because HPLC has high reproducibility, high resolution, and can be detected in part per million levels. Moreover, another advantage of this research study is the clearly results that included text, tables and figures which were not required lengthy and complex explanation.

Minor Project

Minor Project

My research question is concentrated on quantitative analysis of gossypol and its enantiomers acquired from Hibiscus tiliaceus leaves. Gossypol was first found in cottonseed, it could be extracted from several genera that are closely related to the Gossypium. However, an investigation of gossypol enantiomeric contents in leaves of H. tiliaceus has not been studied.

Researchers who have looked at this subject are Jerzy W. Jaroszewski and Kongkiat Pongrod. The arguments were based upon the presence of gossypol in H. tiliaceus.

Jaroszewski et al. (1991) argued that gossypol in H. tiliaceus wood and bark could not be detected by their high performance liquid chromatography (HPLC) method. In addition, they claimed that their HPLC method could separate two enantiomers of gossypol. However, they neither showed the gossypol content nor demonstrate the validity of the method.

Pongrod et al. (2000) argued that gossypol could be isolated from H. tiliaceus by using brine shrimp assay guided fractionation. Moreover, their results were harmonized with the information suggested by the World Health Organization. However, they did not quantitate the gossypol and its enantiomeric contents.

Debate centers on the issue of the presence or absence of gossypol in H. tiliaceus leaves in which two reports provided inconclusive data. There is still work to be done on developing suitable method to quantify gossypol enantiomers and total gossypol content in H. tiliaceus leaves.

My research is closer to Jaroszewski’s work in which HPLC was used to quantify both gossypol enantiomers in H. tilaceus leaves that collected from different sources throughout Thailand.

Hopefully, my contribution to the debate will be served the validated HPLC method for the purpose of screening of gossypol enantiomers and the accurate identification given quality control of the plant material which is an essential prerequisite for ensuring the quality, safety, and efficacy of herbal materials.

References:

Jaroszewski, J., Strøm-Hansen, T., Hansen, S., Thastrup, O., & Kofod, H. (1992). On the botanical distribution of chiral forms of gossypol. Planta Medica, 58(05), 454-458.

Pongrod, K., & Chavasiri, W. (2000). Chemical constituents of the leaves of Hibiscus tiliaceus Linn and their biological activities. In 26th Congress on Science & Technology of Thailand (p. 150). Bangkok: Technical Information Services.

Assignment 2: Writing Introduction

Assignment 2: Writing Introduction

Stage 1:

Medicinal plants have been used to treat sickness throughout human history. The various chemical compounds synthesized from plant are used to support biological function and to prevent from fungi, insects, and herbivorous animals. 

Stage 2:

Cotton plant (Gossypium hirsute, Malvaceae) is an interesting plant which has plenty benefits.  Fiber and seed oil are by-product from cotton industrial process. The mainly active substance from the cotton plant, a yellow flavonoid-liked pigment,  is the polyphenolic compound called “gossypol” (1,1’,6,6’,7,7’-hexahydroxy-5,5’diisopropyl-3.3’dimethyl-[2,2’]binaphthalenyl-[8,8’]dicarbaldehyde). Gossypol has various biological activities such as antiviral, antiprotozoal, anticancer (Dowd et al., 2008). Moreover, Wang et al. (1987) discovered that gossypol showed significant male contraceptive activity via inhibited maturation of human sperm. Qian et al. (1984) and Colin-Negrete et al. (1996) found that gossypol absorption was toxic to animal and human health. In addition, Gilbert et al. (1995) reported that gossypol and gossypolone showed the potent activity of decrease in the number of carcinoma cell in in vivo study. Furthermore, Ford et al. (1991) stated that the (-)-enantiomer was more active in anticancer activity than (+)-enantiomer. Thus, scientists attempted to separate these two isomers. Development of methods to quantitate the gossypol component in cotton has been suggested in various publications. A range of high-performance liquid chromatography (HPLC) methods for separating gossypol enantiomers have been developed since 1968. Cass et al. (1991) analyzed the gossypol isomer ratios in cottonseed samples that were collected from different places. They neither quantitated their results nor demonstrate the validity of the method. However, (R)-(-)-2-amino-1-propanol was used in the method performed by Wu et al. (1988) and Kim et al. (1996). (R)-(-)-2-amino-1-propanol was used to convert gossypol to imine product and developed to analyze samples from plasma and tissue. Nevertheless, their methods were suitable for pharmacokinetic investigation but were not proper for determination of gossypol isomers in cottonseed samples. According to Hron et al. (1999), the (+)- and (-)-gossypol enantiomers separation by high performance liquid chromatography was successful using R-(-)-2-amino-1-propanol as derivatizing substance. This technique becomes a recommended method by the American Oil Chemists’ Society (Ba 8a-99). 

Stage 3:

Although gossypol was first found in cottonseed, but it could be extracted from several genera: Cienfuegosia, Gossypoides, Hampea, Kokia, Lebronnecia, Montezuma, and Thespesia (Jaroszewski J.W., 1991). Hibiscus tiliaceus has long been used in traditional medicines and its genus is close to the Gossypium. However, an investigation of gossypol enantiomers in leaves of H. tiliaceus has not been studied.  

Stage 4 & 5:

The present research aimed at developing a method for quantitation of the gossypol enantiomers and total gossypol content in leaves of Hibiscus tiliaceus collected from different sources throughout Thailand.  The validated HPLC method could be served the purpose of high throughput screening of gossypol enantiomers and the accurate identification given quality control of the plant material is an essential prerequisite for ensuring the quality, safety, and efficacy of herbal materials.

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