Analysis of a Major Phenolic Glucoside and Biochemistry Compounds from Curculigo Orchioides Gaertn

Background: Orcinol-β-D-glucoside, which is also known as orcinol glucoside, is a major phenolic glucoside compound in the rhizome of the Curculigo orchioides Gaertn. This compound has many medicinal properties such as being antioxidant, immunomodulatory, antiosteoporosis, stress relief, antidepressant, etc. Methods: Determination of reducing sugar content by Bertrand’s method, determination of lipid content by Soxhlet method, determination of vitamin C content by iodine titration, determination of enzyme activity catalase by titration with KMnO4. Quantification of Orcinol-β-D-glucoside was conducted by HPLC analysis. Results: The Orcinol-β-D-glucoside of C. orchioides in Thuy Bang mountain was highest. Besides, the content of reducing sugars, vitamin C, enzyme catalase, and lipids of C. orchioides differed significantly among sites. In which, the reducing sugar and vitamin C of C. orchioides in Ngu Binh mountain was highest. Whereas, enzyme catalase was also highest in Thuy Bang mountain. However, the lipid content of C. orchioides was also highest in Huong Tho mountain. Conclusions: The result will contribute to providing the scientific basis for the selection of breeding, planting, development of C. orchioides in Thua Thien Hue province, as well as other provinces in Vietnam and opening new research directions for applications in the future.


Introduction
Curculigo orchioides Gaertn (C. orchioides) belongs to the family Hypoxidaceae, which is one of the important medicinal plants in Vietnam and is popularly recognized by various names such as Sam cau, Ngai cau, Co noc lan (1)(2)(3). It is often used for traditional medicine in China, India and Southeast Asia, including Vietnam. The rhizomes of this plant were used for treatments of impotence, enuresis, cold sperm, back and knee pains, limb numbness, decline in physical strength, diarrhea. They were also used as a potent immunomodulatory and aphrodisiac, etc (2,4). Besides, C. orchioides was also used for treatments of leukorrhea, anemia, neurasthenia, diuretics, rheumatism, and movement difficulty in Vietnam (3,5).
There was some previous research on the pharmacological effects of this compound have shown that Orcinol-β-D-glucoside have antioxidant effects and might be useful to treat skin aging immunomodulatory, antiosteoporosis, anxiolytic effect, antidepressant and it is potential cytotoxic agent against gastric, colon and hepatoma carcinoma cell lines (6)(7)(8)(9)(10)(11)(12)(13)(14). In Vietnam, the study of Nguyen and Nguyen also showed that the antioxidant ability of pharmaceutical materials was quite high with 66.25% (1). Additionally, there were also several quantitative studies of phenolic glucoside compounds including Orcinol-β-D-glucoside but their quantitative methods are different (15,16). In Vietnam, this was a research project to build a qualitative and quantitative method of Orcinol-β-D-glucoside in rhizomes of C. orchioides but its work only evaluated on some samples collected in a limited number of site (17). While the studies on genetic diversity of C. orchioides populations from Vietnam in 2018 shows that there were 7 provinces (including Lai Chau, Quang Binh, Quang Tri, Thua Thien Hue, Quang Nam, Kon Tum and Lam Dong) with high different genetic diversity, therein, the populations originate from Thua Thien Hue had the highest genetic identity (0.9407) (18).
Further, some studies on C. orchioides focus on breeding by rhizome stem cutting and in vitro in Vietnam (19,20). Thus, researching on Orcinol-β-D-glucoside and biochemistry compound in the rhizomes of C. orchioides in sites of Vietnam is needed.

Sample Collection
C. orchioides was collected in 4 sites in Thua Thien Hue province, Vietnam including Phu Bai mountain of Phu Bai town, Huong Tho Mountain of Huong Tra district, Ngu Binh mountain of Hue city, and Thuy Bang Mountain of Huong Thuy district (Fig. 1). In each study locality, we randomly collected 30 trees at a distribution.

Sample Treatment
Samples collected from the wild are washed. The secondary roots were removed, using only rhizomes for analysis. Next, the samples are

Analysis of biochemistry compounds (reducing sugar, lipid, vitamin C, iodine titration and enzyme activity catalase) of Curculigo orchioides
Determination of reducing sugar content by Bertrand's method (21), determination of lipid content by Soxhlet method, determination of vitamin C content by iodine titration, determination of enzyme activity catalase by titration with KMnO4.

Analysis of Orcinol-β-D-glucoside of Curculigo orchioides
One gram of sample was weighed accurately and with 100mL solvent into a 300mL roundbottomed flask. To investigate the effect of solvents and extraction methods on the results of Orcinol-β-D-glucoside quantification, we use the formulas in Table 1. Then, the extract was transferred to centrifugal tube and centrifuged (6000 rpm) for 20 min. The solution was filtrated through a 0.45 µm cellulose acetate membrane filter before HPLC analysis HPLC Fingerprinting. Using HPLC -LC 20AD (Shimadzu) high performance liquid chromatography system (HPLC InertSustain C18 GL Sciences column). Conditions for running HPLC according to Tu (16). Extracts (10μ l) were analyzed using a phase device: ACN: H2O (10:90, v/v) with HPLC column (250 x 4.6mm; 5 µm), helium carrier gas flow at 0.5 ml-1. UV-VIS detector was detected wavelength of 220 nm. Using standard substances with Orcinol-β-D-glucoside of Solarbio (China) has purity of over 98%. Table 1. Formulas used to investigate the effect of solvents and extraction methods on the results of Orcinol-β-D-glucoside of quantification.

Data processing
The collected data is processed on a computer using the SPSS 20 software.

Effect of solvent and extraction method on quantitative results of Orcinol-β-Dglucoside in HPLC fingerprinting
The results showed that the retention time of Orcinol-β-D-glucoside in all treatments around the 20th minute, indicating that this compound is present in 4 samples ( Table 2, Fig. 3). The results of quantitative analysis of Orcinol-β-D-glucoside in the formulas showed that this substance content was different when using different extraction and solvent methods with statistical significance (p< 0.0001, F= 3.415E+11, df= 4). The Tukey's test indicated that the CT 1 was significantly highest compared with the other treatment (CT-2, CT-3), except CT4 (control). Therefore, we use 100% methanol solvent to extract Orcinol-β-D-glucoside from the sample base (Table 2).

Constructing a calibration curve and linear regression equation
The linear dependence of the chromatographic peak area on the concentration of Orcinol-β-Dglucoside are shown at Fig. 3. There is a close correlation between peak area and concentration of Orcinol-β-D-glucoside. The coefficient of determination R 2 = 0.99995 is content with condition 0.99 ≤ R 2 ≤ 1 and the bias Δ does not exceed±15% for all concentrations being used to build the standard line. Therefore, the standard curve that was built is acceptable. Lower-case letters in a cell show the results of significant differences between treatments using Tukey's multiple range test (p< 0.05). The values represent the mean (± SE) of three independent experiments. The highest statistically significant mean value was bold. The signal E+11 means 10 11 .

Effect different site on Orcinol-β-D-glucoside content of C. orchioides
The results of quantitative analysis of Orcinolβ-D-glucoside in the rhizome samples of C. orchioides showed that the content of this substance has a difference between the distribution site with statistical significance (p< 0.0001, F= 8529.239, df= 10). Using Tukey's test shown that the content of Orcinolβ-D-glucoside of C. orchioides distributed in Thuy Bang -Thua Thien Hue was highest (Table 3, Fig. 4). The results of quantitative analysis by HPLC in the rhizome samples of C. orchioides in sites showed that the Orcinolβ-D-glucoside content ranges from 1.27±0.0020 to 9.16±0.0189 mg/g.

Analysis of biochemistry compounds of Curculigo orchioides
The results of quantitative determination of reducing sugars, lipids, vitamin C, and catalase activity in rhizomes of C. orchioides in the sites were statistically significantly different ( Table 2). Using Tukey test shows that the reducing sugar and vitamin C of C. orchioides distributed in Ngu Binh mountain was highest, while the highest lipid of C. orchiodes was Huong Tho mountain. The activity of the catalase enzyme of C. orchioides distributed in Thuy Bang mountain was highest (Table 4). Quantitative results of some biochemical parameters in rhizomes of C. orchioides showed a difference between sites.

Discussion
In our study, the results of quantitative analysis by HPLC in the rhizome samples of C. orchioides in four sites showed that the Orcinolβ-D-glucoside content ranges from 1.27 to 9.16 mg/g. This result was higher than that of He et al (16), in which Orcinol-β-D-glucoside content only ranges from 0.333 to 2.854 mg/g. When using 100% methanol solvent and Soxhlet extraction method, Orcinol-β-Dglucoside quantitative result was higher than when using 70% ethanol solvent and extraction method under reduced pressure at 60 ºC.
This result was similarly to the study of Bian et al. in which the Orcinol-β-D-glucoside content ranges from 1.43 to 11.58 mg/g (15). The samples collected at Thuy Bang mountain (9.16±0.0189 mg/g) were only lower than Yulin -Guangxi (11.58 mg/g) but higher than the 9 remaining sites in China. In comparing this result with the research results in Vietnam, Orcinol-β-D-glucoside content in C. orchioides sample distributed at Thuy Bang mountain was highest (16). This proves that, when using 100% methanol as a solvent, it will yield higher quantitative results than 60% ethanol. From this, we have found that among the sites in Vietnam, C. orchioides distribution in Thuy Bang mountain should be selected for planting and developing and for constructing a product branding later.
Around the world, rhizomes of C. orchioides distribution site was used in making supplements in various types such as tea, powder, extract, and tablet tonic. Besides, the rhizomes extract of C. orchioides distribution was also being used as the main ingredient in the production of cosmetics for women thanks to its anti-inflammatory properties, wound healing, stimulating cell regeneration.
In particular, the reducing sugar content (glucose) in the experimental samples is higher than the results in Joy's study (22). Lipid content in the experimental samples is similar to the results of Joy and Arinathan et al. In particular, the lipid content in the sample of Cucumber originating in Ngu Binh is higher than that of two sites in India (21). The content of vitamin C is much different among sites and lower than the research results of Arinathan et al. (23). The enzyme catalase activity in C. orchioides samples from different origins is very high, proving that the antioxidant capacity of medicinal herbs is very good.
According to Pham et al. when studying the distribution of C. orchioides in Thua Thien Hue, the condition where the distribution of the species is under pine forest with canopy coverage of 0 -50%, distributed in height is from 30 -130 m above water level, the slope is up to 30 ºC, but it is concentrated in the height of 30 -60 m and canopy cover is from 0-5%, temperature is 24 -25.2 ºC, average rainfall year 2,600 -2,800 mm and humidity 83 -84%. In addition, the soil in the natural distribution of C. orchioides is usually brown to reddish brown or pale yellow Leptosols, or Acrisols are gray to grayish yellow, soil mixed with gravel and surface soil often eroded, gravel from 20 -70%; rotten floor mats are very thin and usually only about 0.1 -1.5 cm. In particular, soil moisture in the surface layer is from 0-30 cm quite high, ranging from 30-40% (5).