Isolation of Cottonseed Extracts That Affect Human Cancer Cell Growth

Abstract

Cottonseeds are classified as glanded or glandless seeds depending on the presence or absence of gossypol glands. Glanded cottonseed has anticancer property and glandless cottonseed was reported to cause cancer in one animal study. It is important to investigate the effect of bioactive components from cottonseeds. Our objectives were to isolate ethanol extracts from cottonseeds and investigate their effects on human cancer cells. A protocol was developed for isolating bioactive extracts from seed coat and kernel of glanded and glandless cottonseeds. HPLC-MS analyzed the four ethanol extracts but only quercetin was identified in the glandless seed coat extract. Residual gossypol was detected in the glanded and glandless seed kernel extracts and but only in the glanded seed coat extract. Ethanol extracts were used to treat human cancer cells derived from breast and pancreas followed by MTT assay for cell viability. Ethanol extracts from glanded and glandless cottonseed kernels and gossypol significantly decreased breast cancer cell mitochondrial activity. Ethanol extract from glanded cottonseed kernel and gossypol also significantly decreased pancreas cancer cell mitochondrial activity. These results suggest that ethanol extracts from cottonseeds, like gossypol, contain anticancer activities.

Figure 1

Glanded and glandless cottonseeds and isolation of ethanol extracts from the cottonseeds. (A) Glanded and glandless cottonseeds with short fibers, after sulfuric acid removal of the short fibers, and the seed coat and kernel. Both types of seeds were indistinguishable outside. (B) Section of glanded and glandless cottonseeds. Glanded seeds were smaller than glandless seeds and contained numerous dark green-colored glands in the kernel. (C) Glandless cottonseed coat and kernel. (D) Chloroform extraction. The glanded and glandless kernel homogenates were treated with chloroform followed by centrifugation to separate aqueous (upper) and organic (lower) layers. (E) Hexane extraction: The upper aqueous layer was mixed with hexane followed by centrifugation to separate neutral lipids (upper) and aqueous layers. (F) Ethanol extraction. Seed coat fraction after hexane extraction was suspended in acetic acid, blended, autoclaved and centrifuged. The supernatant was mixed with ethanol followed by centrifugation. The defatted kernel material was directly mixed with ethanol, vortexed and centrifuged. This supernatant was dried under rotoevaporation until all ethanol evaporated. The dried ethanol extracts were stored at −20 °C freezer.

Figure 2

The protocol for isolating ethanol extracts from cottonseeds. This method was consistent of three steps for seed kernel extraction (fractionation, defatting, and ethanol extraction) and four steps for seed coat extraction (fractionation, defatting, acetic acid extraction, and ethanol extraction). Briefly, cottonseeds were ground in dry and in a buffer. The homogenate was separated successively through No. 10 sieve to retain coarse seed coat and No. 40 sieve to retain fine seed kernel and colored superfine filtrate. The coarse seed coat was washed with water and blended several times until clear seed coat pellet was obtained. The colored superfine filtrate was mixed with water and allowed to stand and centrifuged. The red pellet of fine seed coat was pooled together with the coarse seed coat and suspend in the buffer. The seed kernel was suspended in the same buffer followed by grinding and centrifugation. The seed coat suspension and kernel supernatant were defatted with chloroform and hexane followed by centrifugation. Defatted seed coat pellet was air-died after defatting and grinded into fine powder under liquid nitrogen. The fine powder was suspended in acetic acid, ground again, autoclaved, and centrifuged. The supernatant was filtrated through glass wool, mixed with ethanol, stirred well, refrigerated overnight and centrifuged. The supernatant was concentrated in a rotovap and residue ethanol was removed by rotoevaporation. The preparation of ethanol extract from the defatted kernel extract was similar to those of defatted coat extract without acetic acid treatment. The dried ethanol extract pellet was reconstituted in DMSO.

Figure 3

HPLC-MS analysis of ethanol extracts from cottonseeds. HPLC-UV-MS analyzed the four ethanol extracts from glanded and glandless cottonseeds using 5 μl injection of a 10% DMSO solution of the extract. (A) glanded cottonseed coat extract, (B) glanded cottonseed kernel extract, (C) glandless cottonseed coat extract, (D) glandless cottonseed kernel extract.

Figure 4

Detection of gossypol in ethanol extracts from cottonseeds. HPLC-UV-MS analyzed the four ethanol extracts from glanded and glandless cottonseeds using 75 μl injection of a 100% DMSO solution of the extract. (A) glanded cottonseed coat extract, (B) glanded cottonseed kernel extract, (C) glandless cottonseed coat extract, (D) glandless cottonseed kernel extract, (E) gossypol stand, (F) LPS. Left panels: chromatogram of gossypol ion mass at 517.22 (M-H)⁻ with retention time of 24.5 min. Right panels: full mass spectrum.

Figure 5

Effect of cottonseed extracts, gossypol, and LPS on human breast cancer cell growth. Human breast cancer cells (MCF7) were treated with various concentrations of cottonseed extracts and chemicals for 2 and 24 h. The cell media were added with MTT assay reagent, and incubated for 2 h before adding MTT solubilization solution, shaken at room temperature overnight. The color density in the wells was recorded at A570. The data represent the mean and standard deviation of three independent samples. Values with different lower case letters compared to the DMSO control (treatment concentration = 0) in the lower part of the Figure are significantly different at p < 0.05 among the various treatment concentrations in 2 or 24-h treatment time. (A) glanded cottonseed coat extract, (B) glanded cottonseed kernel extract, (C) glandless cottonseed coat extract, (D) glandless cottonseed kernel extract, (E) gossypol, (F) LPS.

Figure 6

Effect of cottonseed extracts, gossypol, and LPS on human pancreatic cancer cell growth. Human pancreatic cancer cells (MIA PaCA-2) were treated with various concentrations of cottonseed extracts and chemicals for 2 and 24 h. The cell media were added with MTT assay reagent, and incubated for 2 h before adding MTT solubilization solution, shaken at room temperature overnight. The color density in the wells was recorded at A570. The data represent the mean and standard deviation of three independent samples. Values with different lower case letters compared to the DMSO control (treatment concentration = 0) in the lower part of the Figure are significantly different at p < 0.05 among the various treatment concentrations in 2 or 24-h treatment time. (A) glanded cottonseed coat extract, (B) glanded cottonseed kernel extract, (C) glandless cottonseed coat extract, (D) glandless cottonseed kernel extract, (E) gossypol, (F) LPS.