Efficient Utilization of Plant Resources by Alkaline Extraction

As compared to the studies with hot water extracts of plants, those with alkaline extracts were limited. Both alkaline and hot water extracts from green tea leaf, oolong tea leaf and orange flower were compared for their biological activities. Plant materials were successively extracted first with hot-water and then alkaline solution, or extracted directly with alkaline solution. Viable cell number of HIV-infected and UV-irradiated cells was determined by MTT method. Antibacterial activity against Porphyromonas gingivalis 381 was determined by turbidity assay. Cytochrome P-450 (CYP)3A4 activity was measured by β-hydroxylation of testosterone using human recombinant CYP3A4 (Figure 5). Radical intensity of superoxide and hydroxyl radical was determined by ESR spectroscopy. Alkaline extraction recovered twice as much as dried materials as compared with water extraction. Water extracts showed higher anti-bacterial, CYP3A4 inhibitory and superoxide scavenging activities, whereas alkaline extract showed higher anti-HIV and hydroxyl radical scavenging activity. Both water and alkaline extracts showed comparable anti-UV activity. The present study suggests the usefulness of alkaline extraction for the efficient utilization of the natural resources. Alternative & Integrative Medicine A l t e rn at ive & Int ative Mdici n e


Introduction
We have previously reported that lignin-carbohydrate complex (LCC) fractions prepared by acid precipitation of the alkaline extracts of pine cone, pine seed shell, catuaba bark, cacao husk, cacao mass, Lentinus edodes mycelia potently protected the cells from HIVinfection [selectivity index (SI)=7~311] [1], and from UV irradiation (SI=7.6~ >38.1) [2]. Similarly, crude alkaline extract of the leaves of Sasa senanensis Rehder (SE) showed comparable anti-HIV (SI=36-45) and anti-UV activity (SI=20~39) with LCC fractions [3]. On the other hand, hot-water extracts of a total of 35 Kampo medicines and their constituent plants had much lower anti-HIV (SI=1~8) and anti-UV activity (SI=1~4.4) [4]. This raised a possibility that the use of alkaline extraction is more advantageous than hot-water extraction to obtain higher amounts of anti-HIV and anti-UV substances. However, this possibility has not yet been tested with water and alkaline extracts prepared from the same plant species. To clarify this point, we prepared hot-water extract (Fr. I), alkaline extract of its residue (Fr. II), and total alkaline extract (Fr. III) from green tea leaf (GT), oolong tea leaf (OT) and orange flower (OF) (Figure 1), and compared their anti-HIV, anti-UV, anti-bacterial, cytochrome P-450 (CYP)3A4 inhibitory and radical scavenging activities, together with their compositional analysis with HPLC.

Preparation of water and alkaline extracts
Five g green tea leaf (GT) (Kimpo, Satoen Co. Ltd., Shizuoka, Japan), oolong tea leaf (OT) (Mitsui Norin Co. Ltd., Tokyo, Japan) or orange flower (OF) (Tochimoto Tenkaido Co.,Ltd., Osaka, Japan) were extracted at 80°C for 30 min with 100 ml of water, and filtered through filter paper (No. 5A, Kiriyama glass Co., Tokyo, Japan) ( Figure 1). The filtrate was concentrated and lyophilized to give the water extract (Fr. I: GT-I, OT-I, OF-I) at the yield of 19.3, 15.6 and 45.2%, respectively. The residue was extracted at 80°C for 30 min with 100 ml of 0.15 M NaOH and filtered. The filtrate was neutralized with HCl, concentrated and lyophilized to give the alkaline extract of the residue (Fr. II: GT-II, OT-II, OF-II) at the yield of 34.0, 30.0 and 19.0%, respectively (18.1, 18.1, 9.7%, respectively, after correction for NaCl present in the extracts). Alternatively, GT, OT or OF (3 g) were directly extracted with 100 ml of 0.15 M NaOH, without hot-water extraction to give the alkaline extract (Fr. III: GT-III, OT-III, OF-III) at the yield of 66.1, 56.4 and 65.4%, respectively (47.3, 35.8 and 42.6%, respectively after correction for NaCl present in the extracts). NaCl, present in GT-II, GT-III,  OT-II, OT-III, OF-II and OF-III,

Assay for anti-HIV activity
Human T-cell leukemia virus I (HTLV-I)-bearing CD4-positive human T-cell line, MT-4, was cultured in RPMI-1640 medium supplemented with 10% FBS and infected with HIV-1IIIB at a multiplicity of infection of 0.01. HIV-and mock-infected MT-4 cells (3×10 4 cells/96-microwell) were incubated for 5 days with different concentrations of samples and the relative viable cell number was determined by MTT assay. The concentration that reduced the viable cell number of the uninfected cells by 50% (CC 50 ) and the concentration that increased the viable cell number of the HIV-infected cells to the 50% that of control (mock-infected, untreated) cells (EC 50 ) were determined from the dose-response curve with mock-infected and HIV-infected cells, respectively. The anti-HIV activity was evaluated by the selectivity index (SI), which was calculated using the following equation: SI=CC 50 /EC 50 [5].

Assay of anti-UV activity
Human oral squamous cell carcinoma HSC-2 cells (Riken Cell Bank, Tukuba, Japan) were inoculated into 96-microwell plates (3×10 3 cells/well, 0.1 ml/well) and incubated for 48 hours to allow cell attachment. The culture supernatant was replaced with 100 μl phosphate-buffered saline without calcium and magnesium [PBS(-)] that contained different concentrations of samples in triplicate, placed at 21 cm distance from a UV lamp (wavelength: 253.7 nm) and exposed to UV irradiation (6 J/m 2 /min) for 1 minute. The cells were then incubated for a further 48 hours in DMEM containing 10% FBS to determine the relative viable cell number by the MTT assay. From the dose-response curve, the CC 50 and the concentration that increased the viability of UV-irradiated cells up to 50% that of control cells (EC 50 ) was determined. The SI was determined using the following equation: SI=CC 50 /EC 50 [6,7].

Measurement of CYP3A4 activity
CYP3A4 activity was measured by β-hydroxylation of testosterone using human recombinant CYP3A4 [9,10]. The reaction mixture, containing 200 mM potassium phosphate buffer (pH 7.4), NADPH regenerating system (1.3 mM NADPH, 1.3 mM glucose-6-phosphate, 0.2 U/ml glucose-6-phosphate dehydrogenase, and 3.3 mM MgCl 2 ) along with 0, 10, 30, 100, 300, 600 and 1000 μg/mL of the test samples or vehicle in triplicate and the human recombinant CYP3A4 (16.5 pmol/ ml), was preincubated at 37°C for 5 min. The reaction was started by the addition of 300 μM testosterone substrates. The final volume of the reaction mixture was 250 μl with a final DMSO concentration of 0.5%. The reaction was stopped by the addition of 500 μl ethyl acetate after 15 min. After centrifugation (15,000 g, 5 min), 400 μl of supernatant was collected, dried, and resuspended in 100 μl of methanol. Analyses of the metabolites were performed by HPLC (JASCO PU2089, AS2057,  UV2075 ChromNAV) equipped with TSKgel ODS-120A, 4.6 mm ID×25 cm, 5 µm column (TOSOH, Tokyo, Japan). The mobile phase consisted of 70% methanol and 30% water. The metabolites were separated using an isocratic method at a flow rate of 1.0 ml/min. Quantification of the All values in alkaline extracts were corrected for NaCl present in the extracts.

Statistical treatment
Experimental values are expressed as the mean±standard deviation (SD). Statistical analysis was performed by using Student's t-test. A p-value <0.01 or <0.05 was considered to be significant.

HPLC separation of the hot-water and alkaline extracts
Major components of GT-I were identified as EGC, EGCg, ECg and caffeine (Figure 2A). On the other hand, major components of GT-II and GT-III were identified as gallic acid and caffeine, while EGC, EGCg and ECg disappeared. Elevated background peaks (retention time: 7.5~25 min) suggests the accumulation of numerous degradation products.
Major products of OT-I were gallic acid, EGC, EGCg, ECg and caffeine ( Figure 2B), whereas major components of OT-II and OT-III were gallic acid and caffeine, and many peaks of degradation products. Major peaks of OF-I, OF-II and OF-III were hesperidin and many degradation products ( Figure 2C).

Anti-UV activity
We recently reported that UV irradiation induced non-apoptotic cell death without induction of internucleosomal DNA fragmentation in HSC-2 cells [6]. UV irradiation (6 J/m 2 /min, 1 minute) significantly reduced the viable cell number after 48 hours' incubation. Addition of tea extracts during the UV irradiation protected the cells from UV-induced cell injury. Green tea leaf extracts showed the highest anti-UV activity, regardless of water extraction (GT-I) (SI=>10.8) or alkaline extraction (GT-II, GT-III) (SI=>10.4, >9.6), although its anti-UV activity was approximately 5-times lower than sodium ascorbate (SI=>58.2) (Exp. 2 in Table 1 Table 1). Water extracts of GT and OT were 4 to 5-times more potent than alkaline extracts. OF extracts were much less potent. All extracts did not show any hormetic stimulation (known as growth stimulation at lower concentration ranges [12]), in contrast to alkaline extract of the leaves of Sasa senanensis Rehder (SE) [8].

Radical-scavenging activity
Water extract of green tea leaf (GT-I) most potently scavenged the superoxide anion (detected as DMPO-OOH), generated by HX and XOD reaction (IC 50 =0.00167 mg/ml) (Exp. 5, Table 1). Alkaline extract of green tea leaf (GT-II, GT-III) showed 5-time lower superoxide scavenging activity (IC 50 =0.00815, 0.00916 mg/ml). Water extract of oolong tea leaf (OT-I) showed comparable superoxide scavenging activity with GT-I, and alkaline extract of it was also 5-times less active. Orange flower extracts showed the weakest superoxide scavenging activity, regardless of water or alkaline extraction.
Water extract of green tea leaf (GT-I) scavenged the hydroxyl radical (detected as DMPO-OH), generated by the Fenton reaction (IC 50 =0.154 mg/ml) (Exp. 6, Table 1). Alkaline extracts of green tea leaf (GT-II, GT-III) were slightly more active. Oolong tea leaf extracts showed comparable activity with green tea leaf extracts, whereas orange flower extracts were 7-times less active. However, in all of these cases, alkaline extracts were slightly more activity than water extracts.

Discussion
The present study demonstrated for the first time that alkaline extracts of green tea, oolong tea leaves and orange flower with 0.15 M NaOH consistently gave much higher anti-HIV activity, as compared with water extracts. The low anti-HIV activity of water extracts (GT-I and OT-I) may be due to the presence of gallic acid, EGC, EGCg and ECg, that had essentially no anti-HIV activity (SI<1) [5]. On the other hand, alkaline extracts (GT-II, GT-III, OT-II, OT-III) contained no detectable amount of EGC, EGCg and ECg, but higher amounts of gallic acid and numerous degradation products. Degradation products rather than gallic acid may be involved in the anti-HIV activity induction. We have recently purified the anti-UV substances (SEE-1) from the alkaline extract of Sasa senanensis Rehder, and identified it as p-coumaric acid derivative(s), a lignin precursor, by recycled HPLC and structural analysis with 1 H-NMR, 13 [14]. Further studies are required to identify the active principle(s) of alkaline extracts of GT, OT and OF. In contrast to higher anti-HIV activity, alkaline extracts of GT and OT showed 4 to 5-times lower antibacterial activity, suggesting that lower molecular weight polyphenols may be involved in the anti-bacterial activity induction.
The present study demonstrated that alkaline extracts (GT-II, GT-III, OT-II, OT-III, OF-II, OF-III) inhibited the CYP3A4 activity to much lesser extent than water extracts (GT-I, OT-I, OF-I) (Figure 4). Alkaline extracts thus seem likely to be safer as compared with water extracts, since the latter are expected to enhance the side-effects of CYP3A4-metabolizable drugs that are administered together.
We also found that alkaline extracts scavenged hydroxyl radical more efficiently than water extracts, whereas water extracts were more active in scavenging superoxide. Hydroxyl radical is known to be highly cytotoxic and mutagenic [15,16], and therefore alkaline extract may prevent or reduce the incidence of hydroxyl radical-induced adverse effects.
We have recently found that alkaline extracts of GT, OT and OF more effectively inhibited the COX-2 activity, as compared with corresponding water extract (Fukuda, manuscript in preparation), suggesting their possible anti-inflammatory activity. Recent clinical research demonststrated that alkaline extracts of of Sasa senanensis Rehder leaf and pine cone of Pinus parviflora Sieb et. Zucc significantly improved the conditions of patients infected with lichenoid dysplasia [17] and herpes-simplex virus [18], respectively.
There was a possibility that some chemical entities in the plant might have been transformed or conversed during alkaline extraction. To test this possibility, gallic acid, EGCg, caffeine or hesperidin (insoluble MeOH was mixed with 10 volume of 0.15 M NaOH, and stood at 80°C for 30 min, or at room temperature for overnight, and then subjected to HPLC analysis. We observed that (i) both gallic acid and EGCg were completely degraded and disappeared, (ii) caffeine was significantly, but not completely degraded, and (iii) hesperidin was stable, with its peak height rather increased, possibly due to increasing solubility under either alkaline conditions (data not shown). This observation was apparently inconsistent from the present results that these compounds were relatively stable in the alkaline extract. Alkaline solution increases the extractability of phenolic compounds, which may reduce the acidity of the extract and then enhance the stability. Gallic acid in the alkaline extracts of green tea may be produced from the degradation of catechin gallates such as EGCg or lignin-related compounds.

Conclusion
The present study demonstrates that water extracts showed higher anti-bacterial, CYP3A4 inhibitory and superoxide scavenging activity, whereas alkaline extracts showed higher anti-HIV and hydroxyl radical scavenging activity. Both water and alkaline extracts showed comparable anti-UV activity. Considering that alkaline extraction gave twice as much as dried materials, as compared with water extraction (Figure 1), it is very useful method to effectively utilize the natural resources. Application of the present alkaline extraction to other plant species may hopefully manufacture products that enrich our daily life.