Prerpints.org logo
Preprint
Communication

Silica Gel Chromatographic Methods for Identification, Isolation and Purification of Gossypol Acetic Acids Prepared in Lab

Altmetrics

Downloads

79

Views

36

Comments

0

A peer-reviewed article of this preprint also exists.

supplementary.pdf (618.13KB )

Submitted:

08 May 2024

Posted:

13 May 2024

You are already at the latest version

Alerts
Abstract
Quite a number of cottonseed varieties are existed. The seeds of each variety produce a different amount of gossypol as a natural toxic compound. The increased interest in this natural product and its toxic effects were enhanced the demand for establishing simple silica-gel-based methods for its isolation, purification, and characterization. Historically, silica gel plays a significant role in isolation and purification of many different natural products. Silica gel is variants and can intro-duce a lab, simple tool for tracking a compound in certain plants during its purification process. In this study, gossypol was isolated and purified in the form of yellow crystals as gossypol acetic ac-id. Several methods and tools were used for its characterization including TLC, preparative TLC, silica gel column, UV/IR spectrophotometer and HPLC (Robust spherical silica gel). A compara-tive study between its amounts in both Egyptian and Chinese varieties were performed. Under the experimental condition, the Egyptian cottonseeds contain 8.705 gm/kg, while the Chinese contains 5.395 gm/kg. Thin Layer Chromatography (TLC) had been flourished the science of the phytochemistry and still popular till nowadays and is recommended for fast, accurate, and inex-pensive evaluation and quantification of gossypol. It is recommended to isolate and purify gossy-pol as gossypol acetic acid using optimized combined methods used in this study. Using Silica gel different protocols will assist and facilitate the parallel comparison and save the compact column from any unexpected contaminants during the purification steps. During each step the silica gel itself could be simply removed by paper filtration.
Keywords: 
Subject: Biology and Life Sciences  -   Biology and Biotechnology

1. Introduction

Gossypol as poisonous pigment obtained its name from the cotton plant (Gossypium barbadense) family Malvaceae. Gossypol is a non-lactonic sesquiterpene. It has different physiological and biological activities. It is usually prepared as gossypol acetic acid (GAA). GAA shows a raising interest because of its biological properties such as: anticancer[1], antiviral [2,3], antimicrobial and antifertility properties [4,5,6]. It can inhibit cellular macromolecules [7]. The crude extract of the cottonseed (containing gossypol) can inhibit the formation of crown gall tumor formation in the potato disc bioassay which were induced by Agrobacterium tumefaciens [8], and can inhibit hepatic microsomal benzo(α) pyrene hydroxylation and hydrogen peroxide production in mice treated with lindane [9].
Gossypol has shown a T/C of 150% at 10 mg/Kg in the PS system [10]. It has a unique chemical structure comprises the following formula: 1, 1', 6, 6', 7, 7'-Hexa hydroxy-3,3'-dimethyl- 5, 5'-bis (1-methylethyl) [2, 2'-binaphthalene] -8, 8'-dicarboxaldehyde; 1,1', 6, 6', 7, 7'-hexahydroxy- 5, 5'-diisopropyl-3, 3'-dimethyl[2, 2'-binaphthalene]-8, 8'-dicarboxaldehyde; 2,2'-bis[1, 6, 7-trihydroxy- 3 -methyl- 5 -isopropyl- 8- aldehyde naphthalene]; 2, 2'-bis[8-formyl-1, 6, 7-trihydroxy-5-isopropyl-3-methyl naphthalene]. It has a general structure of C30H30O8, with molecular weight of 518.54 [11]. In case of GAA its general structure is C30H30O8.C2H4O2, with molecular weight of 578.62.
Its toxicity was investigated on different cancer cell lines such as Her2 positive breast cancer cells [15]. It is able to cause up-regulation of somatostatin receptors 2 and 5 in DU-145 prostate cancer cells [16], and cultured murine erythroleukemia cells[17]. It can inhibit the growth of colorectal cancer and hepatocellular carcinoma [18] and the like. It has also been demonstrated that gossypol inhibits proliferating Ehrich ascites tumor cells [10]. (±)-Gossypol induced apoptosis and autophagy in head and neck carcinoma cell lines [19]. Gossypol decreased cell viability and down regulate the expression of some genes in human colon cancer cells [20]. Gossypol was demonstrated to be specific inhibitor of DNA synthesis [12]. Rosenberg et al. (1986) highlighted that gossypol affects nuclear DNA by upregulating DNA replication and mismatched proteins [13]. The mechanism of anticancer activity of gossypol is inducing apoptosis through the suppression of anti-apoptotic proteins of the Bcl-2 family [14].
Gossypol has antiproliferative and antimetastatic effects on MAT-lyla prostate cancer cells. It can be a potential therapeutic agent for androgen independent human prostate carcinoma [21]. Oral gossypol reported to be safely used on an outpatient basis for the treatment of metastatic adrenal cancer [22]. The genotoxic effect of GAA was evaluated by determining the frequency of micronuclei and mitotic index in male mice bone marrow cells in vivo [23]. The rate of DNA degradation by gossypol-Cu+2 complex was found to be the same both in the presence and absence of molecular oxygen [24,25]. It reported as inhibitor for Bcl-2, Bcl-XL, Bcl-W and Mcl-1 [26]. For more details refer to Fulda (2010) and the references within [26]. For that it and its derivatives are promising candidates for cancer treatment [1,26,27,28].
There is an increasing interest in using gossypol as antivirus [32,33,34,35,36,37]. Gossypol has many other important effects, where both racemic mixture and enantiomers of gossypol inhibit replicating human immunodeficiency virus-type 1(HIV-1) [29,30]. The in vitro anti-amoebic effects of gossypol were reported against axenic trophozoites from five Entamoeba histolytica strains [31].
This study is concerned with simplifying different silica gel chromatographic based methods including TLC, preparative TLC, column chromatography, HPLC (Robust spherical silica gel). Other simple analytical methods such as the UV/infrared spectrophotometric analysis are included. Authentic GAA sample was used in the entire study to assist quantifying and characterizing the prepared GAA. The silica gel-based methods and the other used methods are recommended for fast isolation, characterization, and purification. Silica gel different methods gives different choices. In the present study, the used methods ranging from so simple, handy, in-house, and inexpensive to a highly accurate and sophisticated.

2. Materials and Methods

2.1. Cottonseed Sample

Gossypol was extracted and purified as GAA from Egyptian cottonseed. The used seeds were obtained from Alexandria Oil and Soap Company (Alexandria, Egypt).

2.2. Preparation of Seed Material

Three Kilograms of dried cottonseed were grinded in a blender. The grinded material was passed through a coarse sieve to remove greater parts of the lint and hulls. Two kg of the powdered seeds were transferred into a suitable closed clean and dry glass container.

2.3. Extraction with Petroleum Ether/Diethyl Ether

Two kg of the powdered seeds were first extracted with 3 liters of petroleum ether in a shaker (100 rpm) at room temperature. The resulting extract was filtered through filter paper (Whatman No1). The extraction process was repeated five times to remove the oily component of the powdered seeds. The powdered seeds were followed by air dried at room temperature to get rid of any solvent. The oil-free seed material was then extracted with 3 liters of diethyl ether. The extraction step was further carried out until no change in the color of diethyl ether is observed. The filtrates were concentrated under vacuum using rotatory evaporator at 40oC.

2.4. Precipitation of Gossypol as GAA

The concentrated extract obtained from 2 kg sample was transferred into a conical flask, then the mixture treated with 40 ml glacial acetic acid dropwise until pH 3 was obtained. The flask was well closed and placed in a dark static place (for one week), where yellow crystalline precipitate was obtained at the bottom of the flask. The supernatant liquid was discarded and the precipitate was taken and dried in a desiccator [38].

2.5. Purification and Recrystallization

The precipitate was washed 3 times with 1 ml of glacial acetic acid, followed each time with 10 ml petroleum ether. In each time the mixture was filtered through filter paper (Whatman No1) and finally the precipitate was dissolved in 5 ml diethyl ether containing 1 ml glacial acetic acid. The ether was followed by evaporation until the purified crystals began to be readily separated. The crystals were dried in a desiccator at room temperature.
2.6. Detection and Monitoring GAA by TLC
One milligram of each of the crystalline precipitate and reference GAA (Sigma) were separately dissolved in 50μl chloroform in a clean and dry glass container. Each sample was separately spotted at the starting point (baseline) across the chromatoplate [(20x20 cm) coated with 0.5 mm thick of 0.25 silica gel G60 containing 0.6% starch (Merk)]. The chromoplate was then activated. The samples were vertically migrated using solvent system; ether: petroleum ether (1:1) as a running solution. The plate was allowed to dry and the separated spots can be seen under the visible light as yellow spots (Figure 1) [39]. The TLC was used to monitor GAA in the different samples during the preparation and purification steps (Figure 1).

2.7. Preparative TLC for Purification of GAA

Thirty milligrams of the crystalline precipitate were dissolved in 300μl chloroform in two clean and dry (as a duplicate replica) glass container. The solution was separately streaked in the form of a band across the baseline of the silica gel G60 chromatoplate (containing 0.6% starch as above) and then the chromatoplate was processed using the solvent system (ascending development) ether: petroleum ether (1:1).
The plate was air dried, where two separate bands (a and b) appeared under visible light. Another band (C) on the baseline was neglected (as in Figure 1). Images were taken for visualization. Each band was separately collected, and then eluted with ether, till no residue was obtained upon evaporating a small volume of the colorless eluate to dryness on a watch glass (clean and dry). Each eluted band was filtered where the filtrate was concentrated under vacuum to about 2 ml then left to crystallize. Finally, each crystallized material (a and b as in 2.5.) was dried in a desiccator and then tested for the purity of the GAA using each of the spectrophotometer and HPLC (as below).

2.8. UV Spectrophotometer Analysis for the Spectra of GAA

The crystalline precipitate from petroleum ether-diethyl ether was dissolved in 95% ethyl alcohol, and then determined spectrophotometrically. 40 μg of both GAA precipitate and reference sample were diluted each to 3 ml ethyl alcohol and scanned at 500-188nm using quartz cuvette to determine the presence of GAA in the precipitate.[40]. The absorbance of the top of the best clear band (at 440nm) was used to measure GAA spectrophotometrically in the further investigations.

2.9. GAA Standard Curve

Two milligrams of standard GAA (Sigma Co.) were weighed accurately in a clean and dry glass tube, and were then dissolved in 1 ml absolute ethanol. Volumes of 20, 40, 60, 80, 100, 120, 140, 160, and 180 μl of gossypol were used as a stock solution. Zero absorbance was adjusted against absolute ethanol blank at 440 nm. The absorbency of the prepared dilutions was determined with PYEUnicuan Spectrophotometer The absorbance of each preparation and the standard curve was generated.

2.10. Determination of the GAA (Separated by TLC) Using HPLC

Band A and B from TLC chromatoplate were removed and then separately treated by chloroform to dissolve the separated substance. The chloroform mixture was filtered to remove the silica-starch layer. The filtrate was further evaporated to obtain the purified substance in a crystalline form. 1.9 mg of the purified crystals obtained from band A and 1.5 mg of band B were separately dissolved in 10 ml 70% aqueous acetone. Five μl of each were separately injected in HPLC column (Column Art.720023 ET 250/814 NUCLEOSIL® 10 C18 MACHEREY. NAGEL, Japan) to detect GAA. The column contains Robust spherical silica gel, hydrophobic, weakly polar, end-capped phases for RP chromatography. The separation mechanism was based on hydrophobic (van der Waals) and slight residual silanol interactions. The following was the conditions in which the apparatus was adjusted; UV detector (365 nm), at 35oC, a mobile phase 0.15 M phosphoric acid in acetonitrile/water (4:1), and at a flow rate 1.5 ml/min and at a speed of 5 mm/ml.

2.11. Silica Gel Column Isolation and HPLC Quantification of Gossypol from Egyptian and Chinese Cottonseeds

One gram of fine powdered Egyptian and Chinese cottonseeds, each was separately extracted from 10 ml solution of 70% aqueous acetone in clean and dry glass container then incubated with shaking (150 rpm) at 25oC for 6 hrs. The extract was clarified by centrifugation at 4000 rpm where the supernatant was kept in a clean and dry glass container. Silica gel column chromatography was used for further purification (Merk G120 column 15 cm x 1 cm). The 70% aqueous acetone supernatant was allowed to be adsorbed on the silica gel column, and then washed with 100% petroleum ether to remove any impurities. The purified gossypol in the column was then eluted with 100 ml 70% aqueous acetone.

2.12. HPLC Method for Analysis of GAA in Egyptian and Chinese Cottonseeds

One and half μl from the eluent were taken for detecting the gossypol using HPLC (under the same condition as above). Qualitative and quantitative results of GAA in different samples were made by the comparison between the peaks retention time and peak area of the standard solution and sample extract under the same conditions of operation (as above) [41,42,43].

2.13. Melting Point of GAA

Electrically heated melting point apparatus was used to study the melting point of any crystalline substance. A few crystals of the crystalline precipitate were introduced into the melting point tube, and then exposed to an electrical heat. The reading of the thermometer was taken at the point at which the crystalline precipitate melt. The same procedure was used with a reference sample of GAA (Sigma Co.).

2.14. Infrared Spectral Measurements for GAA

Infrared spectra in a range of 600-4000 cm-1 were measured with a Perkin-Elmer Model 1420 spectrometer. The KBr pellets needed for IR analysis were prepared with the precautions recommended by O’Conner et al (1954) [44], to eliminate moisture. Each sample (0.8 mg) was mixed with 300 mg KBr. The mixture was placed in an agate mortar, and after being grinned manually for several minutes the mixture was transferred to the KBr disk press, which was evacuated for 15 minutes (pressure of 11 tons / cm2) [44,45].

3. Results and Discussion

Cottonseed has economic importance as a source of oil and as animal feed. Even the seeds have high oil and nutritive components, unfortunately a natural toxic pigment is existed which named gossypol (related to the plant name (Gossypium barbadense). Thus, removing gossypol or degrading it is an important step [43,46,47,48,49]. So, there is a real need for a fast, cheap, and practical method(s) for its detection, isolation, purification, crystallization, identification, and quantification.
Additionally, there is an increasing interest concerning the different research done on gossypol, GAA and the other related and derived structures. In such a case, sensitive experiments such as in vitro cancer cell line, gossypol must be used in a highly purified form. Gossypol is usually isolated and detected as GAA. There is an interest in the research considering both gossypol and GAA. Simple silica gel methods could assist better purification. That will protect expensive columns from any unexpected impurities. In addition, using the described method as a model for isolating pure compound from plant source. This will assist isolating another interesting natural product, particularly those which could be isolated using solvents and purified using different silica gel methods.
Silica gel can be used in different forms and combinations with other molecules for isolating botanical compounds. One of its unique properties is its resistance to the chemical changes in presence of different solvents. In contrast to the other form of gels such as the gel groups that are used to isolate proteins; it is more stable and can be easily retrieved from column or from the glass plate surface; cleaned dehydrated and reused. Its chemical properties enable its coexistence with simple assistant compounds like the starch which used for preparing TLC. For its simplicity and efficacy, we aimed to select some of the simplest and most direct silica gel-based methods either from the research previously done on gossypol or from the other phytochemistry protocols. The study introduces a straightforward protocol for isolation, purification, and characterization of GAA. Generally, the methods described in the current study were collected based on their simplicity out of some others tested methods [data not shown] and based on our scientific group experiences in the field of phytochemistry.
Cottonseed from Egypt was collected, grained, and sieved to fine particles to enable better extraction. The grinding step is essential in removing debris and facilitates the extraction process. For defatting the seed materials, petroleum ether was used. After several extraction steps with the ether, the seeds were followed by air dried to remove any residual solvent. After that, diethyl ether was used to extract the gossypol. The diethyl ether extract was reduced to concentrate the sample.at 40oC degree which was used during the entire steps to avoid degradation of gossypol. For that the solvent evaporation was done under vacuum.
Simple methods for purification and recrystallization were used. Multiple runs represent washing with petroleum ether followed by filtration and then, re-dissolving in diethyl were conducted. Finally, acetic acid was used for precipitating the gossypol as GAA. For further purification, TLC preparative chromatography was used. TLC (silica gel G60) preparative chromatography ensures an additional purification step to obtain suitable quantity in a short time. Samples were taken and tested for the presence or for the purity of the GAA in any of the above-described steps as in Figure 1a,b.
Acetic acid was used to precipitate the gossypol as GAA. To our knowledge the perception is succeeded after putting the sample in a tightly closed bottle under static condition in a dark chamber. Apparently, darkness was an essential factor in precipitating gossypol as GAA. The GAA was separated on the TLC for two bands, A and B. Another band, C appeared in the bottom of the TLC beside the starting point (was neglected) as in Figure 1b. The concentration of GAA present in the spot A and B after preparative TLC chromatography using HPLC analysis (Figure 4) indicate that spot A contained 17.5% of GAA while spot B contained 1.02% GAA.
The precipitated gossypol as GAA was subjected to a series of washing steps. The GAA which was obtained from the precipitation and recrystallization process was about 2.6 g/kg cottonseed, and it was in the form of brilliant yellow micro crystals (0.26% based on powdered cottonseed). The difference in the calculated amount between the TLC preparation steps and that obtained from the HPLC data (as below) prove the efficacy of the HPLC in accurate determination of the GAA quantity. Meanwhile, during the crystallization, purification/recrystallization steps loss in the quantity is expected. One should differentiate between the quantitative determination of the existed amount which need a sophisticated instrument like the HPLC which need amount in µg (or less) and the need for purifying suitable amount of gossypol (as GAA) to conduct certain experiment.
The silica gel column chromatography is another alternative purification method was used. Even the gossypol is dissolved in 70% aqueous acetone but upon loading the sample in the silica gel column and because of its absorbency, it enables the sample to be trapped and adsorbed in the column. The silica gel column will do more than one function to purify the gossypol. First it will prevent passing any solid existed material. Second it will enable using a concentrated sample. Third it will enable fast washing using minimum amount of 100% petroleum ether. Fourth, the sample could be simply eluted using the same used solvent which used to dissolve the gossypol (70% aqueous acetone).
The HPLC, a method was adjusted to quantify GAA using spectrophotometer. Authentic sample from Sigma was used as reference. GAA spectra were detected using wavelengths in the range of 188 and 500 nm. The best wavelength in the peaks obtained from UV spectra was used (440 nm) as in Figure 2. Standard curve was generated from the authentic sample and the purified gossypol was evaluated [data not shown]. Furthermore, the spectra of the authentic sample were compared with that of the purified GAA and proved to be identical as in Figure 2a,b. The results obtained either from the standard curve or from the spectra prove that we have a well-purified compound. IR analysis was further used to compare the purified GAA with the authentic sample as in Figure 3. The spectra of both are identical.
Preprints 106022 g002
Figure 2. UV spectrum of GAA reference sample (A); Isolated crystals (B).
Figure 2. UV spectrum of GAA reference sample (A); Isolated crystals (B).
Preprints 106022 g002
Preprints 106022 g003
Figure 3. IR spectrum for (1) GAA reference sample; (2) GAA purified samples.
Figure 3. IR spectrum for (1) GAA reference sample; (2) GAA purified samples.
Preprints 106022 g003
The infrared spectrum of gossypol using potassium bromide disk showed the following absorption bands (Figure 3): the broad band of strong intensity at 3500 and at 3420 cm-1-are attributed to OH stretching vibration. Bands of moderate intensity at 2960, 2920 and 2860 cm-1 are ascribed to CH stretching vibration. The band of strong intensity at 1700 cm-1 is because of CHO stretching vibration. Two bands at 1440 and at 1380 cm-1 are because of C-H banding. The band at 1380 cm-1 is because of CH3 symmetrical deformation. The band at 1340 cm-1 is because of OH bend. Bands characteristic for C-OH appeared at 1240 and at 1180 cm-1. Bands below 1120 (1100, 1050, 960, 840, and 700 cm-1 represent the “finger print’’ of GAA.
The melting points of the crystalline substance and the reference sample of GAA were the same. The melting points were 184oC. Even it is a simple experiment but could prove or disprove the identity of two compared samples. For comprising the gossypol obtained from the Egyptian cottonseed and the Chinese cottonseeds, HPLC was used. Silica gel column was used to absorb the gossypol following by a washing step by ether to remove any impurities. Then the gossypol was eluted using diethyl ether. The results indicated that the yield of gossypol from the Egyptian cottonseed is 8.705 gm/kg while the yield produced from the Chinese cottonseed was 5.293 gm/kg.
The methods used for identifying gossypol and GAA were simplified in this study. We recommend the use of the different silica gel methods and the analyses described in this study to be used for evaluating the gossypol and GAA in the cottonseed oil and animal food products. It might be useful to use cottonseed varieties have less gossypol content in one side. In other side high gossypol content cottonseed is more resistance against different infections.
Preprints 106022 g004
Figure 4. HPLC pattern of A-Authentic sample; B-Egyptian GAA sample; C-Chinese cottonseed gossypol; D-Crystalline purified precipitate from TLC spot (A) and E-Crystalline precipitate gossypol from TLC spot (B).
Figure 4. HPLC pattern of A-Authentic sample; B-Egyptian GAA sample; C-Chinese cottonseed gossypol; D-Crystalline purified precipitate from TLC spot (A) and E-Crystalline precipitate gossypol from TLC spot (B).
Preprints 106022 g004

4. Conclusions

The paper summarizes many experiences in different fields. It combines between a real use of the Silica gel to produce gossypol acetic acid in quantity that can assist the research concerning the it. Different kind of silica gel-based methods are used.
Gossypol was extracted, crystallized, and purified as GAA. Simple silica gel methods were used such as TLC preparative TLC, Silica gel column, and simple spectrophotometric assays beside more sophisticated analysis represented by HPLC. This study showed that the use of TLC could obtain purified GAA and observed quantitatively. The main aim of this study is to enable scientists from purifying the gossypol as GAA using simple and efficient tested silica gel-based methods to use it in different experiments. What is the most important issue in the present investigation? It is the up-front method for isolating the gossypol till its characterization and quantification, which could stand alone as a protocol for preparing gossypol as a GAA in lab. This will enhance its investigation against many cancers’ cell lines and other biological interests.

Funding

Institutional support.

Acknowledgments

The authors acknowledge the entire lab chemists in both Biotechnology Department and the Environmental Science Department (Graduate Studies and Research Institute), as well as anyone who might help and support this study.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Zhang, L.; Jiang, H.; Cao, X.; Zhao, H.; Wang, F.; Cui, Y.; Jiang, B. Chiral gossypol derivatives: Evaluation of their anticancer activity and molecular modeling. European Journal of Medicinal Chemistry 2009, 44, 3961-3972. [CrossRef]
  2. Borovskaya, T.G. Preclinical and clinical evidence of safety of antiviral drug with immunomodulatory activity. Serbian Journal of Experimental and Clinical Research 2018, 19, 271-276. [CrossRef]
  3. Borovskaya, T.G. Safety of the russian antiviral drug kagocel. Terapevticheskii arkhiv 2017, 89, 93-99. [CrossRef]
  4. Chang, Q.; Liu, Z.; Ma, W.Z.; Hei, C.C.; Shen, X.S.; Qian, X.J.; Xu, Z.L. Drug synergistic antifertility effect of combined administration of low-dose gossypol with steroid hormones in rats. Chinese medical journal 2011, 124, 1678-1682.
  5. Kalla, N.R.; Gadru, N.; Foo, T.W. Studies on the male antifertility agent gossypol acetic acid vii. Effect of motility stimulated factors on the revival of human spermatozoal motility after gossypol treatment in vitro. Andrologia 2009, 18, 393-397. [CrossRef]
  6. Waites; Wang; Griffin. Gossypol: Reasons for its failure to be accepted as a safe, reversible male antifertility drug. International Journal of Andrology 1998, 21, 8-12.
  7. Lin, J.; Wu, Y.; Yang, D.; Zhao, Y. Induction of apoptosis and antitumor effects of a small molecule inhibitor of bcl-2 and bcl-xl, gossypol acetate, in multiple myeloma in vitro and in vivo. Oncology reports 2013, 30, 731-738. [CrossRef]
  8. Amara, A.A.; El-masry, M.H.; Bogdady, H.H. Plant crude extracts could be the solution: Extracts showing in vivo antitumorigenic activity. Pak. J. Pharm. Sci. (2008, 21, 159-171.
  9. Awny, H.A.; Amara, A.A.; El-Masry, M.H.; Baghdadi, H.H. Effect of 12 plant extract on hepatic microsomal penzo(a) pyrene hydroxylation and hydrogen peroxide production in mice treated with lendane. J. Environmental Nutritional Interaction 1997, 1, 129-142.
  10. Blasko, G.; Cordell, G.A. Recent developments in the chemistry of plant-derived anticancer agents. Academic Press, U.K.: 1988.
  11. Budavari, S.; Maryadele, J.; Neil, O. The merek index. Eleventh edition on encyclopedia of chemicals, drugs and biologicals. (ed. Pp. . . Merck & Co. Inc. Rahway, N. J. U.S.A. : 1989.
  12. Martinez, F.; R., M.; Espinosa, G.T.; Pardo, J.P. The antifirtility agent, gossypol, releases calcium from rat liver mitochondria. Comp. Biochem. Physiol. C. 1993, 104, 165-169.
  13. Rosenberg, L.J.; Adlakha, R.C.; Desai, D.M.; Rao, P.N. Inhibition of DNA polymerase α by gossypol. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 1986, 866, 258-267.
  14. Zeng, Y.; Ma, J.; Xu, L.; Wu, D. Natural product gossypol and its derivatives in precision cancer medicine. Current Medicinal Chemistry 2019, 26, 1849-1873. [CrossRef]
  15. Bulut, G.; Atmaca, H.; Karaca, B. Trastuzumab in combination with at-101 induces cytotoxicity and apoptosis in her2 positive breast cancer cells. Future oncology (London, England) 2020, 16, 4485-4495. [CrossRef]
  16. Degirmenci, M.; Erdogan, A.P.; Bulut, G.; Atmaca, H.; Uzunoglu, S.; Karaca, B.; Karabulut, B.; Uslu, R. Octreotide in combination with at-101 induces cytotoxicity and apoptosis through up-regulation of somatostatin receptors 2 and 5 in du-145 prostate cancer cells. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2016, 37, 4939-4944. [CrossRef]
  17. Majumdar, S.K.; Daly, E.P.; Kleemeyer, K.M.; Daehler, C.C.; Baker, M.A. Genotoxic effects of gossypol acetic acid on cultured murine erythroleukemia cells. Environmental and molecular mutagenesis 1991, 18, 212-219. [CrossRef]
  18. Mayer, M.; Berger, A.; Leischner, C.; Renner, O.; Burkard, M.; Böcker, A.; Noor, S.; Weiland, T.; Weiss, T.S.; Busch, C., et al. Preclinical efficacy and toxicity analysis of the pan-histone deacetylase inhibitor gossypol for the therapy of colorectal cancer or hepatocellular carcinoma. Pharmaceuticals 2022, 15, 438. [CrossRef]
  19. Benvenuto, M.; Mattera, R.; Masuelli, L.; Taffera, G.; Andracchio, O.; Tresoldi, I.; Lido, P.; Giganti, M.G.; Godos, J.; Modesti, A., et al. (±)-gossypol induces apoptosis and autophagy in head and neck carcinoma cell lines and inhibits the growth of transplanted salivary gland cancer cells in balb/<i>c</i> mice. International Journal of Food Sciences and Nutrition 2016, 68, 298-312.
  20. Cao, H.; Sethumadhavan, k.; Cao, F.; Wang, T. Gossypol decreased cell viability and down-regulated the expression of a number of genes in human colon cancer cells. American Chemical Society (ACS): 2021. [CrossRef]
  21. Chang, C.J.; Ghosh, P.K.; Hu., Y.F.; Brueggemeier, R.W.; Lin., Y.C. Antiproliferative and antimetastatic effects of gossypol on dunning prostata cell-bearing copenhagen rats. Res. Commun .Chem. Pathol. Pharmacol 1993, 79, 293-312.
  22. Flack, M.R.; Pyle, R.G.; Mullen, N.M.; Lorenzo, B.; Wu, Y.W.; Knazek, R.A. Oral gossypol in the treatment of metastatic adrenal cancer. . J. Clin. Endocarin. Matab. 1993, 74, 1019-1024. [CrossRef]
  23. Orhan, G.; Ekmekçi, A.; Menev§e, S. The effect of the male contraceptive agent gossypol acetic acid on mouse bone marrow cells in vivo: Micronuclei and mitotic index. Contraception 1993, 47, 377-385. [CrossRef]
  24. Zaidi, R.; Hadi, S.M. Strand scission in DNA by gossypol and cu(ii): Role of cu(i) and oxygen-free radicals. Journal of Biochemical Toxicology 1992, 7, 213-217. [CrossRef]
  25. Zaidi, R.; Hadi, S.M. Complexes involving gossypol, DNA and cu (2). Biochemi .Int. 1992, 28, 1135-1143.
  26. Fulda, S. Modulation of apoptosis by natural products for cancer therapy. Planta medica 2010, 76, 1075-1079. [CrossRef]
  27. Atmaca, H.; Gorumlu, G.; Karaca, B.; Degirmenci, M.; Tunali, D.; Cirak, Y.; Purcu, D.U.; Uzunoglu, S.; Karabulut, B.; Sanli, U.A., et al. Combined gossypol and zoledronic acid treatment results in synergistic induction of cell death and regulates angiogenic molecules in ovarian cancer cells. European Cytokine Network 2009, 20, 121-130. [CrossRef]
  28. Huang, Y.W.; Wang, L.S.; Dowd, M.K.; Wan, P.J.; Lin, Y.C. (-)-gossypol reduces invasiveness in metastatic prostate cancer cells. Anticancer Res 2009, 29, 2179-2188.
  29. Tai-Shun, L.; Schinazi, R.F.; Zhu, J.; Birks, E.; Carbone, R.; Yikang, S.; Kemei, W.; Liang, H.; Prusoff, W.H. Anti-hiv-1 activity and cellular pharmacology of various analogs of gossypol. Biochemical Pharmacology 1993, 46, 251-255. [CrossRef]
  30. Yang, J.; Li, L.-L.; Li, J.-R.; Yang, J.-X.; Zhang, F.; Chen, G.; Yu, R.; Ouyang, W.-J.; Wu, S.-W. Synthesis and biological evaluation of water-soluble derivatives of chiral gossypol as hiv fusion inhibitors targeting gp41. Bioorganic & Medicinal Chemistry Letters 2018, 28, 49-52. [CrossRef]
  31. González-Garza, M.T.; Matlin, S.A.; Mata-Cárdenas, B.D.; Said-Fernández, S. Differential effects of the (+)- and (–)-gossypol enantiomers upon <i>entamoeba histolytica</i> axenic cultures. Journal of Pharmacy and Pharmacology 1993, 45, 144-145.
  32. Lin, T.S.; Schinazi, R.; Griffith, B.P.; August, E.M.; Eriksson, B.F.; Zheng, D.K.; Huang, L.A.; Prusoff, W.H. Selective inhibition of human immunodeficiency virus type 1 replication by the (-) but not the (+) enantiomer of gossypol. Antimicrobial Agents and Chemotherapy 1989, 33, 2149-2151. [CrossRef]
  33. Polsky, B.; Segal, S.J.; Baron, P.A.; Gold, J.W.M.; Ueno, H.; Armstrong, D. Inactivation of human immunodeficiency virus in vitro by gossypol. Contraception 1989, 39, 579-587. [CrossRef]
  34. Radloff, R.J.; Deck, L.M.; Royer, R.E.; Vander Jagt, D.L. Antiviral activities of gossypol and its derivatives against herpes simplex virus type ii. Pharmacological Research Communications 1986, 18, 1063-1073. [CrossRef]
  35. Royer, R.; Mills, R.; Deck, L.; Mertz, G.; Vanderjagt, D. Inhibition of human immunodeficiency virus type i replication by derivatives of gossypol. Pharmacological Research 1991, 24, 407-412. [CrossRef]
  36. Vagabov, R.M.; Barinskii, I.F.; Dushanbieva, S. [comparative study of the effectiveness of the interferon inducers, poly(i) : Poly(c) and gss (gossypol-beta-aminoethylsodium sulfate), in combination with a specific vaccine in experimental infection with the human acute encephalomyelitis virus]. Vopr Virusol 1980, 334-337.
  37. Wichmann, K.; Vaheri, A.; Luukkainen, T. Inhibiting herpes simplex virus type 2 infection in human epithelial cells by gossypol, a potent spermicidal and contraceptive agent. American Journal of Obstetrics and Gynecology 1982, 142, 593-594. [CrossRef]
  38. Carruth, F.E. Contribution to the chemistry of gossypol, the toxic principle of cottonseed. Journal of the American Chemical Society 1918, 40, 647-663.
  39. Dehe, J.; Xiuhong, X.; Haibao, S.; Jinfang, Z. Thin-layer chromatoghraphy of gossypol. YaoxueTongbao 1981, 16, 14.
  40. Frampton, V.L.; Edwards, J.D.; Henze, H.R. The ultraviolet absorption spectrum of gossypol<sup>1</sup>. Journal of the American Chemical Society 1948, 70, 3944-3945.
  41. Stipanovic, R.D.; Altman, D.W.; Begin, D.L.; Greenblatt, G.A.; Benedict, J.H. Terpenoid aldehydes in upland cottons: Analysis by aniline and hplc methods. Journal of Agricultural and Food Chemistry 1988, 36, 509-515. [CrossRef]
  42. Jaroszewski, J.; Strøm-Hansen, T.; Hansen, S.; Thastrup, O.; Kofod, H. On the botanical distribution of chiral forms of gossypol. Planta medica 1992, 58, 454-458. [CrossRef]
  43. Botsoglou, N.A. Liquid chromatographic determination of unbound and acetone-soluble bound gossypol in cottonseed meals and mixed feeds. Journal of AOAC INTERNATIONAL 1992, 75, 815-823. [CrossRef]
  44. O`Conner, R.T.; Von, D.H.; Dufre, E.F.; Brown, L.E.; Pominski, C.H. The infra-red spectra of gossypol. J. Amer. Chem. Soc. 1954, 76, 2368.
  45. Nakanishik. Infra-red absorption spectroscopy. Holden Day Inc., San Francisco, USA.: 1962.
  46. Câmara, A.C.; Gadelha, I.C.; Borges, P.A.; de Paiva, S.A.; Melo, M.M.; Soto-Blanco, B. Toxicity of gossypol from cottonseed cake to sheep ovarian follicles. PloS one 2015, 10, e0143708. [CrossRef]
  47. Fash, R.H. The effect of crude bollie cottonseed oil upon the color of refined oil from stored crude cottonseed oil. Oil & Soap 1934, 11, 106-109. [CrossRef]
  48. Kramer, R.Y.; Garner, D.L.; Ericsson, S.A.; Wesen, D.A.; Downing, T.W.; Redelman, D. The effect of cottonseed components on testicular development in pubescent rams. Veterinary and human toxicology 1991, 33, 11-16.
  49. Tso, W.W.; Lee, C.S. Cottonseed oil as a vaginal contraceptive. Archives of andrology 1982, 8, 11-14. [CrossRef]
Preprints 106022 g001
Figure 1. (a); Different TLC sample showing different amounts of gossypol; acetic acid (samples collected during the purification steps), 1(b); purified samples show 2 bands A and B.
Figure 1. (a); Different TLC sample showing different amounts of gossypol; acetic acid (samples collected during the purification steps), 1(b); purified samples show 2 bands A and B.
Preprints 106022 g001
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Copyright: This open access article is published under a Creative Commons CC BY 4.0 license, which permit the free download, distribution, and reuse, provided that the author and preprint are cited in any reuse.
A Simplified and Environmental Friendly Processing Procedure for Preserving Gastrodia elata with No Significant Change on the Contents of Gastrodin Derivatives and Anti-Oxidation Activity

Ying Liu

et al.

,

2023

Yield, Characterization and Possible Exploitation of Cannabis Sativa L. Roots Grown Under Aeroponics Cultivation

Fabio Ferrini

et al.

,

2021

Phytochemical Profiling and Biological Potential of Prunus dulcis Shell Extracts

Talel Ben khadher

et al.

,

2023

Prerpints.org logo

Preprints.org is a free preprint server supported by MDPI in Basel, Switzerland.

facebook logotwitter logolinkedin logo
MDPI Initiatives
Important Links
Subscribe

Disclaimer

Terms of Use

Privacy Policy

© 2024 MDPI (Basel, Switzerland) unless otherwise stated