(
%
)

=

ESW
2

(
g
)

ESW
1

(
g
)

×
100
⁢

(
%
)

[

Equation
⁢

3

]

- EY (Extraction Yield, %)=Extraction yield
- ESW₁(Non-extracted Sample Weight, g)=Weight of dried centipede grass leaf sample used for hot water extraction.
- ESW₂(Extracted Sample Weight, g)=Weight of centipede grass hot water extract measured after hot water extraction and complete drying at 105° C. for 24 hours

As a result, the extraction yield of the centipede grass hot water extract calculated through the above [Equation 3] was measured to be 9.04%/mL.

3. In Vitro SPF Measurement of Centipede Grass Extract

In order to confirm the UV blocking ability of the centipede grass extract against UVB having a wavelength of 290 to 320 nm, the sun protection factor (SPF) was measured.

The SPF measurement in this experiment referenced the method of RM Sayre et al. (RM Sayre, et al. A comparison of in vivo and in vitro testing of sunscreening formulas. Photochemistry and photobiology, 1979, 29 (3), pp. 559-566). The in vitro SPF measurement equation used in the present disclosure is as shown in [Equation 4] below.

$\begin{matrix} SPF = CF \times \sum_{290}^{320} EE (λ) \times I (λ) \times Abs (λ) & [Equation 4] \end{matrix}$

- CF=Correction Factor=10
- EE(λ)=Erythema effect of solar radiation at each wavelength λ
- I(λ)=Solar intensity at wavelength λ
- Abs(λ)=Absorbance of sample measured at each wavelength λ

Here, the values in Table 1 below were applied to the EE X I value.

TABLE 1

Wavelength (nm)
EE X I (normalized)

290
0.0150

295
0.0817

300
0.2874

305
0.3278

310
0.1864

315
0.0839

320
0.0180

Total
1.0000

SPF measurement of each of the centipede grass extracts obtained in the above 1. was performed as follows.

- {circle around (1)} Each of the centipede grass extracts obtained in the above 1. (1) to 1. (5) was diluted with each solvent so that the centipede grass extracts were finally 10 mg/mL, 5 mg/mL, 2.5 mg/mL, 1 mg/mL, 0.5 mg/mL, and 0.1 mg/mL.
- {circle around (2)} The solution (100 UL) of the above 1) was put in a 96-well black plate, and the absorbance of the extract was measured using a spectrophotometer (Tecan, Mannedorf, Switzerland) at intervals of 5 nm from 230 nm to 400 nm. Each solvent (80% ethanol, 80% methanol, water, and 70% 1,3-butylene glycol) used for extraction in the above 1. (1) to 1. (5) was used as a standard (blank) (corresponds to ‘each solvent’ in Table 2 below).
- {circle around (3)} The in vitro SPF values calculated by substituting the absorbance measured in the above {circle around (2)} into the above [Equation 4] and Table 1 were shown in Table 2 below.

TABLE 2

In vitro SPF measurement values of each centipede grass extract

Concentration (mg/mL)

Extraction

Each

Solvent
10
5
2.5
1
0.5
0.1
solvent
Blank

Ethanol
19.86
12.71
5.58
3.18
1.44
1.30
1.73
1.36

Methanol
17.91
11.43
4.03
2.98
1.31
1.23
1.16
1.36

Hot water
38.05
38.28
37.49
22.04
11.75
1.76
1.27
1.36

Water
21.63
15.36
9.81
4.98
1.98
1.36
1.20
1.36

1,3-butylene
14.20
8.23
4.11
2.86
1.43
1.31
1.27
1.36

glycol

In Table 2, the blank means an empty well. It was confirmed based on the SPF measurement results of the centipede grass extracts obtained in the above 1. that the centipede grass ethanol extract (10 mg/mL) obtained in the above 1. (1) blocks about 94.9% of UVB, the centipede grass methanol extract (10 mg/mL) obtained in the above 1. (2) blocks about 94.4% of UVB, the centipede grass hot water extract (10 mg/mL) obtained in the above 1. (3) blocks about 97.3% of UVB, the centipede grass water extract (10 mg/mL) obtained in the above 1. (4) blocks about 95.4% of UVB, and the centipede grass 1,3-butylene glycol extract (10 mg/mL) obtained in the above 1. (5) blocks about 92.9% of UVB.

As such, it was confirmed that the centipede grass extract showed all excellent UVB blocking effects in various extraction solvents, and in particular, the UVB blocking effect of the centipede grass hot water extract obtained in the above 1. (3) was superior to that of other solvents. FIG. 1 showed the UV wavelength band absorbance measurement results of the centipede grass hot water extract obtained in the above 1. (3).

4. Ingredient Analysis of Centipede Grass Hot Water Extract

The HPLC analysis was performed to analyze the ingredients of the centipede grass hot water extract of the present disclosure. Specifically, 20 μL of the sample solution (centipede grass hot water extract obtained in the above 1. (3)) was injected into an Agilient Technologies 1200 series HPLC, and a mobile phase composed of a 1% formic acid aqueous solution (solution (A) and a 100% methanol (solution B) was used. Fraction components were eluted using the mobile phase in a combined step and a linear gradient of 100:0 (solution A:B) to 50:50 for 30 minutes, followed by a linear gradient of 50:50 to 0:100 for 60 minutes. A flow rate was 0.5 μL/min and the eluted compound was detected at a wavelength of 360 nm.

As a result, as can be seen in FIG. 2, eight single substances of chlorogenic acid, orientin, isoorientin, rhamnosylisoorientin, derhamnosylmaysin, maysin, luteolin, and eremonetin were confirmed in the centipede grass hot water extract obtained in the above 1. (3), and their contents were shown in Table 3 below.

TABLE 3

Chlorogenic

Ingredient
acid
Orientin
Isoorientin
Rhamnosylisoorientin
Derhamnosylmaysin
Maysin
Luteolin
Eremonetin

μg/g
67.6
7.3
54.0
137.5
157.4
107.2
34.1
85.6

*The above Table 3 showed the contents of each ingredient per 1 mg of the extract in μg.

5. Measurement of Total Phenolic Content and Total Flavonoids Content in Centipede Grass Hot Water Extract

(1) Measurement of Total Phenolic Content in Centipede Grass Hot Water Extract

Phenolic compounds collectively refer to flavonoids, anthocyanins, tannins, catechins, isoflavones, lignans, resveratrols, and the like, and are widely distributed in the plant kingdom. A large number of hydroxyl groups (—OH) present in phenols are known to have excellent antioxidant and anti-inflammatory effects because they have the property of easily bonding to various compounds. Therefore, in order to examine the antioxidant efficacy of the centipede grass hot water extract obtained in the above 1. (3), the total phenolic content in the centipede grass hot water extract was measured. The total phenolic content was measured using the principle that a Folin-reagent is reduced by a phenolic compound to develop a molybdenum blue color.

Specifically, the phenolic content of the centipede grass hot water extract was calculated as an equivalent weight based on a standard calibration curve obtained by preparing the standard material at a concentration of 0 to 100 μg/mL using gallic acid and then analyzing in the same way as the centipede grass hot water extract. The centipede grass hot water extract was mixed with a Folin-Ciocalteau's phenol reagent and reacted at room temperature for 1 hour while blocking light. Thereafter, after dispensing the centipede grass hot water extract into each 96-well plate by 200 μL, the absorbance was measured at 765 nm using a spectrophotometer (Tecan, Mannedorf, Switzerland). The total phenolic content eluted from 1 g of dried centipede grass hot water extract was measured based on the gallic acid.