Triterpenoid Saponins and Flavonoid Glycosides from the Flower of Camellia flavida and Their Cytotoxic and α-Glycosidase Inhibitory Activities

Abstract

Camellia flavida var. flavida, commonly known as “Jinhua Tea”, has its flowers and leaves traditionally utilized as tea and functional food sources. However, there is limited knowledge about its bioactive components and their biological activities. This study isolated ten previously unidentified glycoside compounds from the flowers of Camellia flavida, including three oleanane-type triterpenoid saponins (compounds 1–3) and seven flavonoid glycosides (compounds 4–10), collectively named flavidosides A–J. This study assessed the cytotoxicity of these compounds against a panel of human cancer cell lines and their α-glucosidase inhibitory activities. Notably, flavidoside C showed significant cytotoxicity against BEL-7402 and MCF-7 cell lines, with IC₅₀ values of 4.94 ± 0.41 and 1.65 ± 0.39 μM, respectively. Flavidoside H exhibited potent α-glucosidase inhibitory activity, with an IC₅₀ value of 1.17 ± 0.30 mM. These findings underscore the potential of Camellia flavida in the development of functional foods.

1. Introduction

Yellow camellia, belonging to the Theaceae family and referred to as “Jinhua Tea” in China, was designated as a new food source by the State Food and Drug Administration of China in 2010 and was approved as a food ingredient in 2023. Numerous studies have demonstrated its pharmacological benefits, including antibacterial activity [1], potential hepatitis treatment [2], blood sugar reduction [3], antioxidant effects [4], and cancer prevention capabilities [5]. Camellia flavida var. flavida, a distinguished species of yellow camellia, was identified in Chongzuo, Guangxi, in 1982. This species is characterized by its year-round blooming and substantially higher flower yield compared to other species. Due to its high commercial value and potential as a tea and functional food source, its cultivation has expanded significantly in recent years.

While recent studies by our group have focused on the chemical and biological properties of C. flavida leaves [6,7], research on its flowers has not yet been conducted. Consequently, this study investigates the bioactive constituents and biological activities of C. flavida flowers. Preliminary extraction studies revealed that C. flavida exhibited cytotoxic effects against tumor cells. A substantial body of evidence demonstrates that extracts of C. flavida exert inhibitory actions on non-small cell lung cancer [8], hepatocellular carcinoma [9], and colon cancer [10]. However, these studies predominantly employ crude extracts of C. flavida, whereas many modern chemotherapeutic agents are derived from single, purified natural compounds, such as paclitaxel, vincristine, and onychomycotoxin analogs [11]. Consequently, there is a compelling need to isolate and evaluate individual compounds from C. flavida for their antitumor potential; we assessed the cytotoxic activities of individual compounds against cancer cells lines using the MTT assay.

In traditional Chinese medicine, yellow camellia has long been consumed as a health beverage believed to lower blood sugar levels. Notably, the flavonoids of Camellia nitidissima flower extracts have demonstrated in vitro α-glucosidase inhibitory activity with an IC₅₀ value of 45 μg/mL [12]. The activity of α-glucosidase plays a pivotal role in modulating carbohydrate utilization and absorption in the small intestine. The inhibition of α-glucosidase can effectively diminish carbohydrate digestion and absorption, leading to a notable reduction in postprandial blood glucose levels. This, in turn, aids in the management of metabolic disorders, including diabetes and obesity [13]. Although clinical medications, such as acarbose and voglibose, are available for diabetic patients, they are often associated with and accompanied by undesirable side effects, encompassing flatulence, diarrhea, and abdominal discomfort. As a result, numerous reports have underscored the efficacy of safe, natural phytochemical enzyme inhibitors derived from sources like mulberry leaves, guava fruits, and oil tea seeds in mitigating postprandial hyperglycemia. These findings indicate the potential of C. flavida as a functional food component to control blood glucose and alleviate symptoms of diabetes mellitus.

In this research, ten new compounds (Figure 1), including three triterpenoid saponins (1–3) and seven flavonoid glycosides (4–10), along with five previously identified flavonoids (11–15), were isolated from C. flavida. The cytotoxic activities of compounds 1–10 were assessed against six cancer cell lines using the MTT assay, and their α-glucosidase inhibitory activities were also evaluated.

2. Results and Discussion

Compound 1 was isolated as a white powder, and its molecular formula was determined as C₅₅H₉₀O₂₄ based on HR-ESI-MS analysis (Figure S1, m/z 1157.5726, [M + Na]⁺, calcd 1157.5720). The Liebermann–Burchard reaction for compound 1 was positive, indicating its identity as a potential triterpenoid. NMR analysis (

Table 1. ¹H-NMR (600 MHz) data of compounds 1–3 in Pyr-d₅ (δ_H, mult; J in Hz).

No.	1	2	3
1	0.83 m 1.42 m	0.81 m 1.37 m	0.76 m 1.35 m
2	1.68 m 1.97 m	1.71 m 2.06 m	1.72 m 2.05 m
3	3.17 dd (11.7, 4.1)	3.20 dd (11.3, 3.9)	3.17 dd (11.5, 3.8)
5	0.77 br d (12.3)	0.77 br d (11.9)	0.72 br d (11.6)
6	1.33 m 1.53 m	1.34 m 1.54 m	1.31 m 1.50 m
7	2.03 m 2.15 m	2.03 m 2.15 m	1.85–1.97 m
9	1.68 m	1.68 m	1.49, m
11	1.82, m	1.84, m	1.85, m
12	5.45 t (3.4)	5.44 t (3.4)	5.46 t (3.4)
15	4.48 overlapped	4.48 overlapped	5.09 s
16	4.55 d (3.8)	4.54 d (3.7)
18	2.45 dd (13.5, 3.8)	2.44 dd (13.5, 3.8)	2.71 dd, 13.9, 3.4)
19	1.30 overlapped 2.70 dd (13.5, 12.9)	1.29 overlapped 2.70 dd (13.5, 12.9)	1.21 overlapped 1.56 t-like (13.9)
21	1.40 m 2.39 m	1.39 m 2.39 m	1.29 m 1.76 m
22	2.17 m 2.27 m	2.16 m 2.27 m	1.53 overlapped 2.88 br d (13.7)
23	1.12 s	1.13, s	1.10, s
24	1.02 s	1.04, s	1.04, s
25	0.82 s	0.81, s	0.79, s
26	1.05 s	1.04, s	1.13, s
27	1.81 s	1.82, s	1.34, s
28	3.60 d (10.5) 3.79 d (10.5)	3.60, d (10.5) 3.79, d (10.5)	3.81, d (10.8) 4.42, overlapped
29	1.05 s	1.04, s	0.85, s
30	1.10 s	1.10, s	0.89, s
GlcA-1	4.86 overlapped	4.86, overlapped	4.82, d (7.1)
2	4.68 m	4.73, m	4.71, m
3	4.67 m	4.74, m	4.74, m
4	4.52 m	4.64, m	4.63, dd (9.5, 9.0)
5	4.45 overlapped	4.57, m	4.57, d (9.5)
6-OCH3	3.69 s
Glc-1	5.87 d (7.4)	5.90 d (7.4)	5.90 d (7.4)
2	4.10 dd (9.0, 7.4)	4.12 dd (9.3, 7.4)	4.10 dd (9.0, 7.4)
3	4.40 m	4.41 m	4.39 m
4	4.07 t-like (9.1)	4.07 t-like (9.0)	4.04 t-like (9.1)
5	4.40 m	4.41 m	4.39 m
6	4.31 m 4.66 m	4.31 m 4.66 m	4.28 m 4.66 m
Gal-1	6.10 d (7.7)	6.18 d (7.6)	6.21 overlapped
2	4.69 m	4.71 m	4.71 m
3	4.45 m	4.48 m	4.49 br d (9.2)
4	4.45 m	4.46 m	4.44 br s
5	4.23 m	4.26 m	4.27 m
6	4.31 m	4.32 m	4.33 m
Rha-1	6.23 br s	6.23 br s	6.21 br s
2	4.77 br s	4.78 br s	4.76 br s
3	4.71 m	4.72 m	4.69 m
4	4.20 t-like (9.2)	4.21 t-like (9.1)	4.20 t-like (9.1)
5	4.84 m	4.88 m	4.89 m
6	1.41 d (6.1)	1.42 d (5.8)	1.40 d (5.8)

and

Table 2. ¹³C-NMR (151 MHz) data of compounds 1–3 in Pyr-d₅ (δ_C).

No.	1	2	3
1	39.0	39.0	39.0
2	26.4	26.5	26.6
3	89.7	89.8	89.8
4	39.6	39.7	39.7
5	55.6	55.6	55.6
6	18.8	18.9	18.7
7	36.8	36.8	36.1
8	41.4	41.5	41.8
9	47.4	47.3	47.1
10	37.0	37.1	37.0
11	24.0	24.0	24.1
12	123.9	123.9	126.0
13	146.0	146.0	142.0
14	48.0	48.0	54.3
15	67.4	67.4	73.8
16	78.7	78.7	216.1
17	41.1	41.1	53.2
18	43.3	43.3	48.1
19	48.0	48.0	48.0
20	31.3	31.3	31.0
21	37.1	37.1	35.8
22	31.0	31.0	27.1
23	28.1	28.0	28.0
24	16.8	16.8	16.9
25	15.9	15.9	15.8
26	17.6	17.6	17.7
27	21.0	21.0	21.7
28	69.6	69.6	70.9
29	33.5	33.5	33.1
30	24.6	24.6	23.6
GlcA-1	105.2	105.4	105.4
2	79.5	79.5	79.6
3	82.8	82.8	82.7
4	70.9	71.3	71.3
5	76.6	77.4	77.4
6	170.3	172.9	172.2
6-OCH3	52.1
Glc-1	102.9	102.7	102.8
2	76.4	76.4	76.5
3	78.5	78.5	78.5
4	72.6	72.6	72.7
5	78.2	78.1	78.2
6	63.5	63.6	63.7
Gal-1	101.6	101.4	101.4
2	76.2	76.4	76.5
3	76.1	76.0	76.1
4	71.1	71.2	71.3
5	77.0	77.0	77.1
6	62.0	62.0	62.1
Rha-1	102.3	102.4	102.4
2	72.6	72.6	72.7
3	72.6	72.6	72.7
4	73.9	74.0	74.0
5	69.8	69.8	69.9
6	18.3	18.3	18.3

, and Figures S2–S10) revealed seven singlet methyl groups at chemical shifts δ_H 0.82, 1.02, 1.05 × 2, 1.10, 1.12, and 1.81 in the up-field region, and δ_H 2.45 (dd, J = 13.5, 3.8 Hz, H-18), a notable proton signal with two characteristic unsaturated carbons at δ_C 146.0 (C-13) and δ_C 123.9 (C-12), indicating an olean-12-ene pentacyclic triterpenoid skeleton. Additionally, three oxymethine protons were observed at δ_H 3.17, 4.48, and 4.55, corresponding to carbons C-3 (δ_C 89.7), C-16 (δ_C 78.7), and C-15 (δ_C 67.4), respectively. Two methylene protons attached to C-28 (δ_C 69.6) appeared as a doublet at δ_H 3.60 and 3.79 (both d, J = 10.5 Hz). Moreover, HMBC correlations (Figure 2A) indicated three hydroxy groups linked to C-15, C-16, and C-28, demonstrated by HMBC cross-peaks from δ_H 4.48 (H-15) to δ_C 48.0 (C-14), 41.1 (C-17), and 21.0 (C-27), from δ_H 4.55 (H-16) to δ_C 48.0 (C-14) and 41.1 (C-17), and from δ_H 3.60/3.79 (H₂-28) to δ_C 41.1 (C-17) and 31.0 (C-22). The α-orientation of H-3 was confirmed by the trans-diaxial coupling constant of a large ³J value (10.2 Hz) and NOESY correlations (Figure 2B) of δ_H 3.17 (H-3)/0.77 (H-5). The cis-orientations of H-15/H-16 were suggested by a small ³J value of H-16 (3.8 Hz), and NOESY correlations of δ_H 4.48 (H-15)/1.05 (H₃-26)/3.60 (H₂-28), δ_H 4.55 (H-16)/3.60 (H₂-28), and δ_H 3.60 (H₂-28)/2.45 (H-18)/1.05 (H₃-29) established that H-15 and H-16 were β-oriented. Thus, the aglycone part of compound 1 was established as a 3,15,16,28-tetrahydroxyolean-12-ene framework. Moreover, the NMR characteristics were found to be very similar to those of camellianol F [14].

Anomeric proton signals at δ_H 4.86, 5.87, 6.10, and 6.23 were observed and displayed HSQC correlations with the anomeric carbon signals at δ_C 105.2, 102.9, 101.6, and 102.3, respectively, characteristic of the four monosaccharide units. The ¹³C-NMR signals of the four sugars were determined from the HMBC and TOCSY spectra. Specifically, δ_C 105.2, 79.5, 82.8, 70.9, 76.6, and 170.3 were assigned to glucopyranuronic acid. δ_C 102.9, 76.4, 78.5, 72.6, 78.2, and 63.5 were associated with glucopyranose. δ_C 101.6, 76.2, 76.1, 71.1, 77.0, and 62.0 were identified as galactopyranose, and δ_C 102.3, 72.6, 72.6, 73.9, 69.8, and 18.3 were attributed to rhamnopyranose. Additionally, acid hydrolysis of 1 yielded D-glucuronic acid, L-rhamnose, D-glucose, and D-galactopy, analyzed by HPLC.

Further analysis of the HMBC correlations of δ_H 3.69 (GlcA-6-OCH₃) and 4.45 (GlcA-H-5) to δ_C 170.3 (GlcA-C-6) indicated a methyl ester group was present at GlcA-C-5. The β-configuration of glucopyranosyl and galactopyranosyl units was confirmed by the relatively large coupling constants (J = 7.4 and 7.7 Hz) of their anomeric protons. The broad singlet of its anomeric proton confirmed the α-configuration of the rhamnopyranosyl unit. The β-configuration of the 6-methyl glucopyranuronoyl unit was indicated by the NOESY correlations of δ_H 4.86 (GlcA-H-1)/4.67 (GlcA-H-3)/4.45 (GlcA-H-5).

The HMBC correlations from Gal-H-1 (δ_H 6.10) to GlcA-C-3 (δ_C 70.9) and from Glc-H-1 (δ_H 5.87) to GlcA-C-2 (δ_C 79.5) suggested that the GlcA was linked to Gal-C-1 and Glc-C-1 via GlcA-C-2 and GlcA-C-3, respectively. The rhamnose was connected to Gal-C-2, as evidenced by the HMBC correlation from Rha-H-1 (δ_H 6.23) to Gal-C-2 (δ_C 76.2). The HMBC correlation from GlcA-H-1 (δ_H 4.86) to C-3 (δ_C 89.7) of the aglycone unit confirmed that the tetrasaccharide moiety was located at C-3 of the triterpene skeleton. Therefore, the structure of 1 was established as 3β,15α,16α,28-tetrahydroxyolean-12-en-3-O-β-D-glucopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→3)]-β-D-glucuronopyranoside methyl ester, assigned the trivial name flavidoside A.

Compound 2 was obtained as a white powder and displayed a molecular formula of C₅₄H₈₈O₂₄ by HR-ESI-MS (Figure S11, m/z 1143.5554, [M + Na]⁺, calcd 1143.5564). The NMR data (Table 1 and Table 2, and Figures S12–S19) for 2 were very similar to 1. Compounds 1 and 2 shared the same glycoside moiety, specifically camellianol F. However, they differ in the type of sugar moiety that was attached to this common glycoside. The HSQC correlations from δ_H 4.86 to δ_C 105.4, from δ_H 5.90 to δ_C 102.7, from δ_H 6.18 to δ_C 101.4, and from δ_H 6.23 to δ_C 102.4 suggested the presence of four sugar units. According to other NMR spectra and acid hydrolysis followed by HPLC analysis, compound 2 comprised β-D-glucopyranuronic acid (GlcA), β-D-glucopyranosyl (Glc), α-L-rhamnopyranosyl (Rha), and β-D-galactopyranosyl (Gal) sugar units. GlcA was linked to Gal-C-1 and Glc-C-1 via GlcA-C-2 and C-3, respectively, and rhamnose was connected to Gal-C-2, as evidenced by the HMBC correlations (Figure S20) from δ_H 6.18 (Gal-H-1) to δ_C 82.8 (GlcA-C-3), from δ_H 5.90 (Glc-H-1) to δ_C 79.5 (GlcA-C-2), and from δ_H 6.23 (Rha-H-1) to δ_C 76.4 (Gal-C-2). Thus, the structure of compound 2 was established as 3β,15α,16α,28-tetrahydroxyolean-12-en-3-O-β-D-glucopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→3)]-β-D-glucuronopyranoside (flavidoside B).

Compound 3 was isolated as a white powder. The molecular formula was deduced as C₅₄H₈₆O₂₄ by HR-ESI-MS (Figure S21, m/z 1141.5389 [M + Na]⁺, calcd 1141.5407). The ¹H and ¹³C NMR data were very similar to those of compound 2, except for the absence of the hydroxy group at C-16, which was replaced by a carbonyl group in the triterpene skeleton. The sugar chain was identical in compounds 3 and 2, as confirmed by the NMR data (Table 1 and Table 2, and Figures S22–S31). As a result, compound 3 was elucidated as 3β,15α,28-trihydroxyolean-12-en-16-on-3-O-β-D-glucopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→2)-β-D-galactopyranosyl-(1→3)]-β-D-glucuronopyranoside (flavidoside C).

Compound 4 was isolated as a yellow amorphous powder. The molecular formula was determined to be C₃₃H₄₂O₁₉ from its positive HR-ESI-MS (Figure S32, m/z 765.2207 [M + Na]⁺, calcd 765.2218). δ_H 5.92 (H-6) and 5.90 (H-8) in the ¹H-NMR spectrum (

Table 3. ¹H (600 MHz) and ¹³C (151 MHz) NMR data of compound 4 in CD₃OD.

No.	δH (J in Hz)	δC	No.	δH (J in Hz)	δC
2	5.19 d (10.4)	83.5	4	3.29 overlapped	73.9
3	4.55 d (10.4)	81.2	5	4.18 m	70.9
4		195.7	6	1.20 d (6.2)	17.9
5		165.5	Glc-1	4.02 d (7.6)	105.3
6	5.92 d (1.9)	97.4	2	3.30 overlapped	78.9
7		168.6	3	3.41 t-like (9.1)	78.3
8	5.90 d (1.9)	96.3	4	3.33 overlapped	70.9
9		164.0	5	3.10 dt (9.5, 3.1)	77.2
10		102.5	6	3.68 d (3.1)	62.1
1′		128.7	Rha2-1	5.21 d (1.5)	101.8
2′, 6′	7.36 d (8.4)	130.1	2	3.88 dd (3.3, 1.5)	71.9
3′, 5′	6.85 d (8.4)	116.5	3	3.79 dd (9.5, 3.3)	71.8
4′		159.2	4	3.33 overlapped	74.2
Rha1-1	4.71 d (1.4)	102.1	5	4.02 m	70.3
2	3.46 dd (3.2, 1.4)	82.4	6	1.26 d (6.2)	17.9
3	3.72 dd (9.8, 3.2)	71.6

and Figures S33–S40) displayed a pair of doublets (J = 1.9 Hz) representing aromatic protons. The aromatic proton signals at δ_H 6.85 (2H, d, J = 8.4 Hz) and 7.36 (2H, d, J = 8.4 Hz) of the A₂B₂ type suggested a 1,4-disubstituted B-ring. Two oxygenated methines were observed at δ_H 4.55 and 5.48 (both d, J = 10.4 Hz). These findings indicated that compound 4 was a 3,5,7,4′-tetrahydroxyflavanone (dihydrokaempferol) [15].

Compound 4 contained three sugar moieties, deduced from the anomeric signals of δ_H/C 4.71/102.1, 4.02/105.3, and 5.21/101.8. The ¹³C-NMR, HMBC, and TOCSY spectra displayed the signal attribution of three sugars: δ_C 102.1, 82.4, 71.6, 73.9, and 70.9 were assigned to one rhamnopyranose; δ_C 101.8, 71.9, 71.8, 74.2, 70.3, and 17.9 were associated with another rhamnopyranose; δ_C 105.3, 78.9, 78.3, 70.9, 77.2, and 62.1 were identified as the glucopyranose. Moreover, the configurations of L-rhamnose and D-glucose were determined by acid hydrolysis and HPLC identification. The anomeric proton at δ_H 4.02 (d, J = 7.6 Hz) suggested the β-configuration of glucopyranosyl. The two α-configuration units of rhamnopyranosyl were determined by the broad singlet of their anomeric protons. Thus, compound 4 was identified as a 3,5,7,4′-tetrahydroxyflavanone substituted with two α-L-rhamnopyranosyl moieties and a β-D-glucopyranosyl moiety.

The linkage of the rhamnopyranosyl unit at C-2 of the glucopyranosyl unit and the glucopyranosyl moiety was attached to C-2 of another rhamnopyranosyl moiety, confirmed by the HMBC correlations (Figure 3) from δ_H 5.21 (Rha2-H-1) to δ_C 78.9 (Glc-C-2) and from δ_H 4.02 (Glc-H-1) to δ_C 82.4 (Rha1-C-2). Moreover, the oligosaccharide chain was linked at C-3 of the aglycone, also confirmed by the HMBC correlation from δ_H 4.71 (Rha1-H-1) to δ_C 81.2 (C-3) and the reverse correlation from δ_H 4.55 (H-3) to Rha1-C-1 (δ_C 102.1).

The large ³J_H-2/H-3 value (10.4 Hz) hinted at the trans-form orientation of H-2 and H-3. The ECD spectrum of 4 (Figure 4) showed a positive Cotton effect at 330 nm (Δε +2.19) and a strong negative Cotton effect at 292 nm (Δε −9.50), establishing that the aglycone of 4 was 2R and 3R [16]; this was confirmed by the calculated ECD of 4 as well. Based on the above analysis, compound 4 could be assigned as (2R,3R)-3,5,7,4′-tetrahydroxyflavanone-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-α-L-rhamnoside (flavidoside D).

Compound 5 was isolated as a yellow powder with the molecular formula determined to be C₃₅H₄₄O₂₀ using HR-ESI-MS data (Figure S41, m/z 807.2327, [M + Na]⁺, calcd 807.2324). The ¹H and ¹³C NMR data (

Table 4. ¹H (600 MHz) and ¹³C (151 MHz) NMR data of compounds 5 and 6.

No.	5 1		6 2
	δH (J in Hz)	δC	δH (J in Hz)	δC
2	5.48 d (2.3)	82.1	5.42 d (8.4)	80.9
3	4.17 d (2.3)	75.8	4.71 d (8.4)	76.9
4		194.4		193.8
5		166.1		163.4
6	5.93 d (1.7)	97.5	5.90 s	96.0
7		168.9		167.1
8	5.97 d (1.7)	96.3	5.90 s	95.1
9		164.6		161.9
10		101.9		101.0
1′		128.0		126.2
2′, 6′	7.31 d (8.5)	129.0	7.25 d (8.4)	128.8
3′, 5′	6.82 d (8.5)	116.3	6.78 d (8.4)	115.3
4′		158.7		157.7
5-OH			11.71 br s
7-OH			10.96 br s
4′-OH			9.62 br s
Rha1-1	5.36 d (1.2)	99.6	4.77 br s	99.7
2	3.73 br s	81.8	3.49 br s	77.9
3	3.53 dd (9.7 3.4)	71.6	3.09 m	70.1
4	3.18 t-like (9.7)	73.4	3.15 m	72.1
5	2.38 dq (9.7 6.1)	70.3	3.60 m	69.0
6	0.88 d (6.1)	17.9	1.07 d (6.0)	17.6
Glc-1	4.47 d (7.7)	105.5	4.31 d (7.8)	102.5
2	3.41 overlapped	78.4	3.17 m	75.6
3	3.46 t-like (9.0)	78.7	3.28 m	77.5
4	3.39 overlapped	71.1	3.46 m	70.0
5	3.20 m	77.4	3.07 m	76.6
6	3.74 m 3.85 m	62.4	3.48 m 3.64 m	60.8
Rha2-1	5.27 d (1.8)	101.3	5.18 br s	99.5
2	4.00 dd (3.2 1.8)	69.8	3.70 br s	70.3
3	5.03 dd (9.8 3.2)	75.5	3.75 m	67.7
4	3.50 t-like (9.8)	71.6	4.72 t-like (9.8)	74.1
5	4.19 m	70.1	4.07 m	65.7
6	1.24 d (6.2)	17.9	0.94 d (6.1)	17.4
Acetyl-1		172.8		170.2
2	2.08 s	21.1	2.00 s	21.0

¹ Measured in CD₃OD. ² Measured in DMSO-d₆.

, Figures S42–S49) for 5 were similar to 4, with an added acetyl moiety at position Rha2-C-3, suggested by the HMBC correlations (Figure 5) from δ_H 5.03 (Rha2-H-3) and 2.08 (acetyl-H-2) to δ_C 172.8 (acetyl-C-1). The small ³J_H-2/H-3 value (2.3 Hz) hinted at the cis-form orientation of H-2/H-3. The absolute configuration of 2R, 3S was confirmed by comparing the experimental ECD and calculated ECD data of 5 (Figure 6). Thus, compound 5 was assigned as (2R,3S)-3,5,7,4′-tetrahydroxyflavanone-3-O-[3-acetyl-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)]-α-L-rhamnoside (flavidoside E).

Compound 6 was obtained as a yellow solid, with the same molecular formula as 5 (C₃₅H₄₄O₂₀) deduced by HR-ESI-MS (Figure S50, m/z 807.2311 [M + Na]⁺, calcd 807.2324). The ¹H and ¹³C NMR data (Table 4, Figures S51–S57) were very similar to compound 5 except for the position of the acetyl unit. The acetyl unit linked to C-4 of the rhamnopyranosyl moiety was confirmed by the HMBC correlation (Figure S58) from δ_H 4.72 (Rha2-H-4) to δ_C 170.2 (acetyl-C-1). The large ³J_H-2/H-3 value (8.4 Hz) revealed the trans-form orientation of H-2/H-3 and the absolute configuration of 2R,3R, which was displayed by the ECD spectra (Figure S59). Thus, compound 6 was identified as (2R,3R)-3,5,7,4′-tetrahydroxyflavanone-3-O-[4-acetyl-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)]-α-L-rhamnoside (flavidoside F).

Compound 7 was a yellow powder and displayed the molecular formula of C₄₆H₅₂O₂₆ (Figure S60, m/z 1021.2837, [M + H]⁺, calcd 1021.2825) based on the HR-ESI-MS data. The presence of 3,5,7,3′,4′-pentahydroxyflavone aglycone (quercetin) [17] was confirmed by a pair of aromatic proton signals at δ_H 6.13 and 6.32 (both br s) along with 6.88 (d, J = 7.8 Hz), 7.55 (br d, J = 7.8 Hz), and 7.60 (1H, br s), as well as the HMBC correlations (Figure 7) from δ_H 7.60 (H-2′) and 7.55 (H-6′) to δ_C 158.9 (C-2) in the ¹H-NMR spectrum (

Table 5. ¹H-NMR (600 MHz) data (J in Hz) of compounds 7–10.

No.	7 1	8 2	9 1	10 1
6	6.13 br s	6.15 br s	6.17 br s	6.15 br s
8	6.32 br s	6.33 br s	6.35 br s	6.35 br s
2′	7.60 br s	7.51 overlapped	7.98 d (8.3)	7.98 d (8.6)
3′			6.89 d (8.3)	6.90 d (8.6)
5′	6.88 d (7.8)	6.84 d (8.8)	6.89 d (8.3)	6.90 d (8.6)
6′	7.55 br d (7.8)	7.51 overlapped	7.98 d (8.3)	7.98 d (8.6)
5-OH		12.58 br s
Glc1-1	5.58 d (7.8)	5.57 d (7.9)	5.53 d (8.0)	5.57 d (8.0)
2	5.24 dd (9.5, 7.8)	5.09 dd (9.5, 7.9)	5.20 dd (9.5, 8.0)	5.19 dd (9.5, 8.0)
3	3.88 m	3.82 t-like (9.0)	3.90 t-like (9.2)	3.87 t-like (9.0)
4	3.48 m	3.29 m	3.43 m	3.42 t-like (9.2)
5	3.58 m	3.52 m	3.52 m	3.54 m
6	3.51 m 3.88 m	3.41 overlapped 3.73 br d (11.3)	3.47 m 3.86 m	3.47 m 3.87 m
Ara-1	4.34 d (6.8)			4.34 d (7.0)
2	3.54 m			3.53 m
3	3.49 m			3.47 m
4	3.77 br s			3.77 br s
5	3.56 m 3.85 m			3.56 m 3.87 m
Glc2-1		4.31 d (7.9)	4.42 d (7.7)
2		2.95 m	3.18 dd (9.0, 7.7)
3		3.20 m	3.28 m
4		3.26 m	3.27 m
5		3.10 m	3.32 m
6		3.41 overlapped 3.68 m	3.62 dd (11.6, 6.3) 3.86 m
Rha-1	4.58 br s	4.38 br s	4.54 br s	4.54 d (1.5)
2	3.85 br s	3.57 br s	3.80 br s	3.81 dd (3.1, 1.5)
3	3.60 dd (9.2, 3.0)	3.32 m	3.55 dd (9.3, 3.1)	3.56 m
4	3.44 t-like (9.4)	3.26 m	3.42 t-like (9.4)	3.43 t-like (9.4)
5	3.53 m	3.32 m	3.49 m	3.51 m
6	1.13 d (6.0)	0.96 d (6.0)	1.11 d (6.1)	1.12 d (6.1)
Xyl-1	4.39 d (7.4)	4.24 d (7.5)	4.35 d (7.5)	4.36 d (7.5)
2	3.26 dd (9.1, 7.4)	3.02 m	3.26 dd (9.0, 7.5)	3.25 dd (9.1, 7.5)
3	3.37 t-like, (9.0)	3.12 m	3.35 t-like (9.0)	3.35 t-like (9.0)
4	3.49 m	3.03 m	3.49 m	3.49 m
5	3.17 t-like (10.5) 3.80 m	3.00 m 3.60 dd (11.1, 5.0)	3.15 t-like (10.7) 3.79 m	3.15 t-like (10.8) 3.79 m
Coumaroyl-2, 6	7.45 d (8.0)	7.51 d (8.4)	7.47 d (8.2)	7.46 d (8.2)
3, 5	6.80 d (8.0)	6.79 d (8.4)	6.81 d (8.2)	6.80 d (8.2)
7	7.68 d (15.9)	7.54 d (15.9)	7.70 d (15.9)	7.69 d (15.9)
8	6.42 d (15.9)	6.36 d (15.9)	6.39 d (15.9)	6.39 d (15.9)

¹ Measured in CD₃OD. ² Measured in DMSO-d₆.

).

The 1D and 2D NMR spectra (Figures S61–S67) of 7 suggested that it contains four sugar units: glucopyranose (δ_C 100.7, 74.5, 84.5, 70.3, 76.8, 68.4), arabinopyranose (δ_C 105.2, 72.2, 73.9, 69.5, 67.2), rhamnopyranose (δ_C 102.2, 71.6, 82.4, 72.7, 69.5, 17.9), and xylopyranose (δ_C 106.2, 75.3, 77.5, 71.1, 66.8). Acid hydrolysis and HPLC confirmed the configuration of these sugars.

HMBC correlations were observed from δ_H 3.88 (Glc-H-3) to δ_C 105.2 (Ara-C-1) and from δ_H 3.51 (Glc-H-6) to δ_C 102.2 (Rha-C-1), establishing that Glc is linked to Ara-C-1 and Rha-C-1 through Glc-C-2 and Glc-C-6, respectively. The xylopyranoyl unit was linked to Rha-C-3, as shown by the HMBC correlation from δ_H 4.39 (Xyl-H-1) to δ_C 82.4 (Rha-C-3). The sugar chain was positioned at C-3 of the flavone, confirmed by the HMBC correlation from δ_H 5.58 (Glc-H-1) to δ_C 134.8 (C-3). Signals for a 1,4-disubstituted benzene ring proton at δ_H 6.80 (2H, Coumaroyl-H-2 and 6) and 7.45 (2H, Coumaroyl-H-3 and 5), and for trans-oriented vinylic protons at δ_H 6.42 (Coumaroyl-H-8) and 7.68 (Coumaroyl-H-7), were noted. Moreover, HMBC correlations from δ_H 6.42 (Coumaroyl-H-8) to δ_C 127.3 (Coumaroyl-C-1), 168.8 (Coumaroyl-C-9), from δ_H 7.68 (Coumaroyl-H-7) to δ_C 131.3 (Coumaroyl-C-2 and Coumaroyl-C-6), and 115.1 (Coumaroyl-C-8) suggested a p-E-coumaroyl unit. The HMBC correlation from δ_H 5.24 (Glc-H-2) to δ_C 168.8 (Coumaroyl-C-9) indicated that coumaroyl was attached to Glc-C-2. Therefore, compound 7 was identified as 3,5,7,3′,4′-pentahydroxyflavonol-3-O-α-L-arabinopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-2-O-p-E-coumaryl-β-D-glucopyranoside, named flavidoside G.

Compound 8, isolated as a yellow solid, had its molecular formula determined as C₄₇H₅₄O₂₇ from the ion peak at m/z 1073.2747 [M + Na]⁺ (calcd 1073.2750, Figure S68) in the HR-ESI-MS. The ¹H- NMR spectrum (Table 5) showed that 8 contained the same flavone aglucone structure of quercetin as 7. The difference between 8 and 7 was in the sugar units. The sugar chain in 8 included two β-D-glucopyranosyl units, a α-L-rhamnopyranosyl unit, and a β-D-xylopyranoyl unit, as suggested by the 1D and 2D NMR data (Figures S69–S75) and acid hydrolysis. HMBC correlations (Figure S76) from δ_H 4.24 (Xyl-H-1) to δ_C 81.1 (Rha-C-3), from δ_H 4.38 (Rha-H-1) to δ_C 67.9 (Glc1-C-6), from δ_H 4.31 (Glc2-H-1) to δ_C 83.1 (Glc1-C-3), from δ_H 5.09 (Glc1-H-2) to δ_C 165.7 (Coumaroyl-C-9), and from δ_H 5.57 (Glc1-H-1) to δ_C 132.8 (C-3) revealed the positions of the sugars and confirmed that the sugar chain was located at C-3 of the aglucone. Based on this evidence, the structure of 8 was identified as 3,5,7,3′,4′-pentahydroxyflavonol-3-O-β-D-glucopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-2-p-E-coumaryl-β-D-glucopyranoside (flavidoside H).

Compound 9, obtained as a yellow powder, had its molecular formula identified as C₄₇H₅₄O₂₆ by HR-ESI-MS (Figure S77, m/z 1035.3014, [M + H]⁺, calcd 1035.2981). The ¹H and ¹³C NMR data (Table 5 and

Table 6. ¹³C-NMR (151 MHz) data of compounds 7–10 (δ_C).

No.	7 1	8 2	9 1	10 1
2	158.9	156.8	159.1	159.1
3	134.8	132.8	134.6	134.7
4	178.9	177.0	179.0	179.0
5	162.9	161.2	163.0	163.0
6	99.9	98.6	99.9	99.9
7	165.5	164.1	165.6	165.6
8	94.8	93.6	94.9	94.9
9	158.3	156.4	158.4	158.5
10	105.9	104.0	105.8	105.9
1′	123.3	120.9	122.8	122.9
2′	117.5	116.3	132.3	132.3
3′	145.8	144.8	116.2	116.2
4′	149.6	148.6	161.3	161.3
5′	116.2	115.2	116.2	116.2
6′	123.5	121.7	132.3	132.3
Glc1-1	100.7	98.8	100.7	100.7
2	74.5	72.2	74.4	74.5
3	84.5	83.1	84.6	84.3
4	70.3	69.2	70.4	70.4
5	76.8	75.3	76.9	76.9
6	68.4	67.9	68.6	68.5
Ara-1	105.2			105.3
2	72.2			72.2
3	73.9			73.9
4	69.5			69.5
5	67.2			67.2
Glc2-1		103.3	104.8
2		73.2	74.7
3		76.8	77.6
4		69.5	71.3
5		76.4	78.0
6		61.0	62.5
Rha-1	102.2	101.3	102.2	102.2
2	71.6	69.8	71.7	71.7
3	82.4	81.1	82.4	82.4
4	72.7	70.7	72.6	72.7
5	69.5	68.0	69.5	69.5
6	17.9	17.6	17.9	17.9
Xyl-1	106.2	105.4	106.3	106.3
2	75.3	73.9	75.2	75.3
3	77.5	76.0	77.5	77.5
4	71.1	69.9	71.0	71.1
5	66.8	65.5	66.8	66.8
Coumaroyl-1	127.3	125.3	127.3	127.4
2, 6	131.3	130.2	131.3	131.3
3, 5	116.8	115.8	116.8	116.8
4	161.1	159.7	161.2	161.2
7	147.3	144.6	147.2	147.3
8	115.1	114.4	115.2	115.2
9	168.8	165.7	168.6	168.6

¹ Measured in CD₃OD. ² Measured in DMSO-d₆.

, and Figures S78–S86) were similar to those of compound 8. The distinction of 9 was its 1,4-disubstituted B-ring, evidenced by its A₂B₂-type aromatic proton signals at δ_H 6.89 and 7.98 (both d, J = 8.3 Hz). The flavone aglucone structure of 9 was kaempferol [18]. Thus, compound 9 was designated as 3,5,7,4′-tetrahydroxyflavonol-3-O-β-D-glucopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-2-p-E-coumaryl-β-D-glucopyranoside (flavidoside I).

Compound 10 was obtained as a yellow powder. Its molecular formula was determined to be C₄₆H₅₂O₂₅ (Figure S87, m/z 1005.2885, [M + H]⁺, calcd 1005.2876) based on HR-ESI-MS. Its ¹H and ¹³C NMR data (Table 5 and Table 6, and Figures S88–S96) were very similar to those of compound 7. The difference in 10 was the presence of para substitution on the B-ring. Thus, compound 10 was designated as 3,5,7,4′-tetrahydroxyflavonol-3-O-α-L-arabinopyranosyl-(1→3)-[β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)]-2-p-E-coumaryl-β-D-glucopyranoside (flavidoside J).

The known compounds 11 to 15 were identified as pratensein-7-O-β-D-glucoside (11) [19], quercetagetin-7-O-β-D-glucoside (12) [20], quercetin-3-O-β-D-glucoside (13) [21] apigenin-8-C-α-L-arabinosyl-6-C-β-D-glucoside (14) [22], and 3,4′-,5,7-tetrahydroxy-3′-methoxyflavone-3-O-β-D-glucopyranoside (15) [23], respectively (Figures S97–S106).

The cytotoxicity of compounds 1–10 was evaluated against six human cancer cell lines and two normal human cell lines using the MTT method. Compounds 1–3, representing newly identified triterpenoid saponins, exhibited inhibitory effects against Hela, MCF-7, BEL-7402, Hep G2, and MDA-MB-231 cell lines. Notably, compound 3 demonstrated potent cytotoxic effects against the MCF-7 and BEL-7402 cell lines, surpassing the positive control with IC₅₀ values of 1.65 ± 0.39 and 4.94 ± 0.41 μM, respectively. Moreover, its inhibitory effect on MDA-MB-231 cells was comparable to that of cisplatin. As a novel oleanane-type saponin analog, compound 3 fell within a class of triterpenoid saponins renowned for their potent antitumor properties, positioning it as a promising lead candidate for antitumor drug development [24]. Notably, the IC₅₀ value of compound 3 against MCF-7 cells (1.65 ± 0.39 µM) was significantly lower than the reported range (6.1~16.0 µM) for oleanane-type saponins in the study by Huyen, L.T. et al. [25], highlighting its enhanced potency. A thorough literature review on the cytotoxicity of C. flavida revealed a lack of prior studies investigating the effects of its active components on BEL-7402 cells. Our findings contribute to bridging this knowledge gap and underscore the potential of compound 3 in the treatment of breast and liver cancer.

Compound 2 demonstrated significantly greater cytostatic effects on Hela cells compared to the positive control, with an IC₅₀ value of 4.17 ± 0.85 μM. In contrast, compound 3, along with compound 1, exhibited less pronounced inhibitory effects. A review of the literature suggested that the n-butanol and water-soluble fractions of the ethanolic extract from C. flavida were more potent in inhibiting Hela cell proliferation. Quantitative analysis identifies saponins as the primary constituents in these fractions [26]. Consequently, compound 2 from C. flavida emerged as a promising lead candidate for cervical cancer treatment.

In contrast, all flavonoid saponins (4 to 10) exhibited only weak cytotoxicity against the six cancer cell lines. The triterpenoid saponins exhibited significantly stronger cytotoxicity than the flavonoid glycosides across all six cancer cells. Moreover, none of the compounds displayed cytotoxicity towards normal cells (

Table 7. Cytotoxicity (IC₅₀, μM ± SD, n = 8) of compounds 1–10 against cell lines.

	Hela	MCF-7	BEL-7402	A549	Hep G2	MB-231	MRC-5	HNEpC
1	15.07 ± 1.06	9.06 ± 0.45	7.62 ± 1.06	>30	25.60 ± 3.46	>30	>30	>30
2	4.17 ± 0.85	19.24 ± 3.71	>30	>30	14.04 ± 3.29	26.53 ± 3.07	>30	>30
3	>30	1.65 ± 0.39	4.94 ± 0.41	23.42 ± 7.26	8.77 ± 1.56	10.08 ± 2.28	>30	>30
4	>30	>30	>30	>30	>30	>30	>30	>30
5	>30	>30	>30	>30	20.76 ± 3.79	>30	>30	>30
6	>30	>30	11.40 ± 3.11	>30	>30	>30	>30	>30
7	>30	>30	>30	>30	>30	>30	>30	>30
8	>30	>30	>30	>30	>30	>30	>30	>30
9	24.72 ± 4.39	>30	>30	>30	28.80 ± 5.55	>30	>30	>30
10	>30	>30	>30	17.38 ± 2.81	>30	>30	>30	>30
PC	11.06 ± 0.72 1	3.79 ± 0.26 2	9.94 ± 0.80 1	4.20 ± 0.37 1	6.31 ± 0.18 1	9.06 ± 1.01 1	ND	ND

Positive control (PC): ¹ cisplatin and ² doxorubicin.

).

Despite the cytotoxicity results of compounds 4–10 being unsatisfactory, the flavonoid glycosides exhibited strong α-glucosidase inhibitory activity. In contrast, none of the triterpenoid saponins exhibited any inhibitory effect on α-glucosidase. The inhibitory activity of compounds 7 and 10 towards α-glucosidase was equivalent to that of acarbose, exhibiting IC₅₀ values of 2.38 ± 0.22 mM and 2.26 ± 0.48 mM, respectively. Compound 9, on the other hand, demonstrated a moderate level of inhibitory activity against α-glucosidase, with an IC₅₀ value of 3.33 ± 0.48 mM. Compound 8 demonstrated strong inhibitory activity against α-glucosidase, with an IC₅₀ value of 1.17 ± 0.30 mM, which was superior to that of acarbose (2.04 ± 0.27 mM). The initial analysis of SAR (Structure-Activity Relationship) revealed that the presence of a double bond at positions C-2 and C-3 in the parent nucleus diminished the inhibitory effect on α-glycosidase. Additionally, the number of hydroxyl groups on the skeleton and the type of monosaccharide in the sugar chain had minimal impact on this inhibitory effect. Notably, the introduction of the p-E-coumaroyl group significantly enhanced the inhibitory effect of the compound against α-glycosidase. Accordingly, we could hypothesize that flavonoid glycosides 4–10 derived from the water fraction of C. flavida constitute a significant basis for its hypoglycemic activity (

Table 8. α-Glucosidase inhibition activity (IC₅₀, mM ± SD, n = 3) of compounds 1–10.

	IC50		IC50
1	>20	7	2.38 ± 0.22
2	>20	8	1.17 ± 0.30
3	>20	9	3.33 ± 0.48
4	6.15 ± 0.43	10	2.26 ± 0.48
5	12.73 ± 1.08	acarbose	2.04 ± 0.27
6	8.09 ± 2.61

).

To explore the reasons behind the inhibitory effects of compounds 1 to 10 on α-glucosidase, molecular analysis docking was conducted to uncover the potential mechanism (Figure 8 and Figures S107–S115). Among these, compound 8 exhibited a binding affinity of −5.48 kcal/mol and effectively occupied the active site pockets, indicating multiple binding interactions. A hydrogen bond was formed between the C-8-OH group and residue A307 at a distance of 2.9 Å. Additionally, two hydrogen bonds were identified between the carbonyl group at C-4 and the C-5-OH group, both with residue A307 at distances of 3.5 Å and 3.1 Å, respectively. The Glc-3-OH group simultaneously formed two hydrogen bonds with residues T310 and A307 at distances of 2.9 Å and 3.2 Å, respectively (Table S1).

3. Experimental Procedures

3.1. General Experimental Procedures

UV spectra were obtained using a UNICO UV-2802 Spectrometer (Shanghai UNICO Instruments Co., Ltd., Shanghai, China). ECD spectra were measured at room temperature on a Jasco J-815 CD spectrometer (JASCO Corporation, Tokyo, Japan). IR spectra were collected using a Shimadzu IRAffinity-1S FTIR spectrometer (Shimadzu Corporation, Tokyo, Japan). NMR spectra were acquired on Bruker AVANCE III HD 600 MHz spectrometers (Bruker Corporation, Billerica, MA, USA) with Pyr-d₅ (δ_H 7.20, 7.57, 8.72 and δ_C 123.44, 135.43, 149.84), CD₃OD (δ_H 3.31 and δ_C 49.00), and DMSO-d₆ (δ_H 2.50 and δ_C 39.52) as solvents. HR-ESI-MS data were collected on a Waters G2-XS Q-TOF mass spectrometer (Waters Corporation, Milford, MA, USA) equipped with a BEH-C18 column. Column chromatography was performed using Toyopearl Butyl 650C (Nippon Chemi-Con Corporation, Tokyo, Japan), MCI GEL (CHP20, Mitsubishi Chemical Corporation, Tokyo, Japan), Toyopearl HW-40F (Nippon Chemi-Con Corporation, Tokyo, Japan), macroporous resin of D101-type (Tianjin Berens Biotechnology Co., Ltd., Tianjin, China), and C18 reverse-phase silica gel (SMB 100-20/45, Fuji Silysia Chemical Ltd., Tokyo, Japan). Semipreparative HPLC was conducted with a Welch XB-C18 column (5 μm, 10 × 250 mm, 4 mL/min, Shanghai Welch Technology Co., Ltd., Shanghai, China) on a Laballiance HPLC system (LabAlliance, State College, PA, USA) with a Model 500 UV detector. Analytical HPLC was performed using an SSI HPLC system (Scientific Systems, Inc., State College, PA, USA) with a Welch XB-C18 column (5 μm, 4.6 × 250 mm, 1.0 mL/min) and a Model 201 UV detector. Medium-pressure liquid chromatography was conducted on a Büchi B-688 chromatography pump (BÜCHI Labortechnik Aktiengesellschaft, Flawil, Switzerland) with a C-635 UV photometer.

3.2. Plant Material

The flowers of C. flavida were authenticated and provided by Guangxi Fangcheng Golden Camellias National Nature Reserve Management Centre in January 2022. A voucher specimen (No. 20220201-8126) of the C. flavida plant was deposited in the Medical School of Guangxi University.

3.3. Extraction and Isolation

Air-dried flowers of C. flavida (2.5 kg) were extracted with 70% EtOH (9 L) for 7 days, yielding a concentrated extract (1 kg). This extract was diluted with water and extracted sequentially with petroleum ether and ethyl acetate. The aqueous layer was filtered and subjected to chromatography on D101 macroporous resin, eluted with an EtOH/H₂O gradient (0:1, 1:9, 3:7, 5:5, 7:3, and 9:1), resulting in 5 fractions (Fr.1–Fr.5). Fraction Fr.3 (130 g, 50% EtOH) was further separated on MCI resin with a MeOH/H₂O gradient (0:100 to 95:5), yielding 12 sub-fractions (Fr.3.1–Fr.3.12). Sub-fraction Fr.3.8, eluted with 80% MeOH, was subjected to medium-pressure liquid chromatography on an ODS column, eluted with a MeOH/H₂O gradient (30% to 100% MeOH), and produced 9 sub-fractions (Fr.3.8.1–Fr.3.8.9). Fr.3.8.9 was further chromatographed on a Butyl 650C column, eluted with a MeOH/H₂O gradient (10% to 100% MeOH), isolating compound 5 (15.1 mg) and 6 (19.0 mg). Fr.3.8.7 was purified using semi-preparative with CH₃CN/0.1% TFA-H₂O (35:65, v/v) under isocratic conditions, yielding compounds 1 (t_R = 20.57 min, 60.3 mg), 2 (t_R = 12.53 min, 11.4 mg), and 3 (t_R = 18.45 min, 201.4 mg). The 90% EtOH-eluted fraction (Fr.10) was separated using medium-pressure liquid chromatography on a C18 column, eluted with a MeOH/H₂O gradient (50% to 100% MeOH), producing 7 sub-fractions (Fr.3.10.1–Fr.3.10.7). Fr.3.10.1 was further chromatographed on HW-40F resin with an EtOH/H₂O gradient (0:1, 1:9, 3:7, 5:5, 7:3, 9:1), yielding compounds 7 (47.2 mg) and 10 (9.4 mg). Fr.3.10.2 was purified using a semi-preparative Welch XB-C18 column with CH₃CN/0.1% TFA-H₂O (18:82, v/v), yielding compounds 8 (t_R = 22.02 min, 161.7 mg), 9 (t_R = 41.95 min, 22.8 mg), and 15 (t_R = 95.82 min, 30.2 mg). Finally, Fr.3.12 (20 g) was separated on MCI resin with a MeOH/H₂O gradient (from 0:100 to 95:5), affording 7 fractions (Fr.3.12.1–Fr.3.12.7). Fr.3.12.7 (4155 mg) was chromatographed on the Butyl 650C, eluted with a MeOH/H₂O gradient (10% to 100% MeOH), isolating compounds 11 (427.3 mg) and 14 (52.7 mg). Meanwhile, Fr.3.12.6 (6074 mg) was purified using a semi-preparative Welch XB-C18 column with CH₃CN/0.1% TFA-H₂O (56:44, v/v), yielding compounds 4 (t_R = 45.37 min, 12.8 mg), 12 (t_R = 69.08 min, 55.4 mg), and 13 (t_R = 95.44 min, 333.1 mg).

3.3.1. Compound 1

Flavidoside A (1): Appears as a white amorphous powder; [α]²⁵_D +25.9 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 204 (1.06); IR (ATR) v_max 3402, 2945, 1750, 1304, 1080 cm⁻¹; NMR spectra recorded in Pyr-d₅, refer to Table 1 and Table 2; HR-ESI-MS m/z 1157.5726 [M + Na]⁺ (calcd for C₅₅H₉₀O₂₄Na⁺, 1157.5720).

3.3.2. Compound 2

Flavidoside B (2): Appears as a white amorphous powder; [α]²⁵_D +40.1 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 211 (1.35); IR (ATR) v_max 3390, 2933, 1742, 1295, 1081 cm⁻¹; NMR spectra recorded in Pyr-d₅, refer to Table 1 and Table 2; HR-ESI-MS m/z 1143.5554 [M + Na]⁺ (calcd for C₅₄H₈₈O₂₄Na⁺, 1143.5564).

3.3.3. Compound 3

Flavidoside C (3): Appears as a white powder; [α]²⁵_D +84.6 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 200 (0.92); IR (ATR) v_max 3382, 2921, 1751, 1294, 1083 cm⁻¹; NMR spectra recorded in Pyr-d₅, refer to Table 1 and Table 2; HR-ESI-MS m/z 1141.5389 [M + Na]⁺ (calcd for C₅₄H₈₆O₂₄Na⁺, 1141.5407).

3.3.4. Compound 4

Flavidoside D (4): Appears as a yellow amorphous powder; [α]²⁵_D −19.5 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 196 (2.03), 236 (0.96), 290 (0.76); IR (ATR) v_max 3375, 1716, 1625 cm⁻¹; ECD (c 1.0 mg/mL, CH₃OH) λ_max (Δε) 254 (0.81), 292 (−10.50), 330 (2.09) nm; ¹H-NMR and ¹³C-NMR in CD₃OD, refer to Table 3; HR-ESI-MS m/z 765.2207 [M + Na]⁺ (calcd for C₃₃H₄₂O₁₉Na⁺, 765.2218).

3.3.5. Compound 5

Flavidoside E (5): Appears as a yellow amorphous powder; [α]²⁵_D −28.6 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 190 (1.82), 223 (1.07), 294 (0.66); IR (ATR) v_max 3380, 1721, 1633 cm⁻¹; ECD (c 1.0 mg/mL, CH₃OH) λ_max (Δε) 260 (1.07), 300 (−8.71), 350 (2.44) nm; ¹H-NMR and ¹³C-NMR in CD₃OD, see Table 4; HR-ESI-MS m/z 807.2327 [M + Na]⁺ (calcd for C₃₅H₄₄O₂₀Na⁺, 807.2324).

3.3.6. Compound 6

Flavidoside F (6): Appears as a yellow solid; [α]²⁵_D −16.3 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 191 (1.09), 220 (061), 294 (0.28); ECD (c 1.0 mg/mL, CH₃OH) λ_max (log ε) 252 (0.94), 292 (-5.84), 334 (1.96); IR (ATR) v_max 3385, 1708, 1637 cm⁻¹; ¹H-NMR and ¹³C-NMR in DMSO-d₆, see Table 4; HR-ESI-MS m/z 807.2311 [M + Na]⁺ (calcd for C₃₅H₄₄O₂₀Na⁺, 807.2324).

3.3.7. Compound 7

Flavidoside G (7): Appears as a yellow powder; [α]²⁵_D −85.9 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 209 (2.06), 251 (1.62), 270 (1.44), 310 (1.73), 358 (1.70); IR (ATR) v_max 3380, 1702, 1640, 1442, 1165 cm⁻¹; ¹H-NMR and ¹³C-NMR in CD₃OD, see Table 5 and Table 6; HR-ESI-MS m/z 1021.2837 [M + H]⁺ (calcd for C₄₆H₅₃O₂₆⁺, 1021.2825).

3.3.8. Compound 8

Flavidoside H (8): Appears as a yellow solid; [α]²⁵_D −111.2 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 204 (1.85), 255 (150), 281 (1.29), 310 (1.42), 350 (1.51); IR (ATR) v_max 3395, 1711, 1651, 1444, 1160 cm⁻¹; ¹H-NMR and ¹³C-NMR in DMSO-d₆, see Table 5 and Table 6; HR-ESI-MS m/z 1073.2747 [M + Na]⁺ (calcd for C₄₇H₅₄O₂₇Na⁺, 1073.2750).

3.3.9. Compound 9

Flavidoside I (9): Appears as a yellow powder; [α]²⁵_D −71.4 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 200 (2.52), 244 (2.01), 277 (1.85), 304 (2.09), 366 (1.97); IR (ATR) v_max 3383, 1708, 1643, 1450, 1162 cm⁻¹; ¹H-NMR and ¹³C-NMR in CD₃OD, see Table 5 and Table 6; HR-ESI-MS m/z 1035.3014 [M + H]⁺ (calcd for C₄₇H₅₅O₂₆⁺, 1035.2981).

3.3.10. Compound 10

Flavidoside J (10): Appears as a yellow powder; [α]²⁵_D −42.6 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 201 (1.47), 262 (1.08), 282 (0.96), 305 (0.82), 364 (0.94); IR (ATR) v_max 3395, 1713, 1640, 1436, 1159 cm⁻¹; ¹H-NMR and ¹³C-NMR in CD₃OD, see Table 5 and Table 6; HR-ESI-MS m/z 1005.2885 [M + H]⁺ (calcd for C₄₆H₅₃O₂₅⁺, 1005.2876).

3.3.11. Purity Statement

The purity of all compounds was verified to be ≥95% by HPLC analysis.

3.4. Acid Hydrolysis

Acid hydrolysis and the identification of monosaccharides followed the method described in the previous literature [27].

3.5. ECD Calculation

ECD calculations were performed using the method described in a previous paper from our laboratory [7].

3.6. Cytotoxicity Assays

3.6.1. Cell Culture and Treatment

The human cervical cancer cell line Hela, the human lung adenocarcinoma cell line A549, the human breast cancer cell lines MCF-7 and MDA-MB-231, the human hepatoma cell lines BEL-7402 and Hep G2, the human embryonic lung fibroblast cell line MRC-5, and the human nasal epithelial cell line HNEpC were all sourced from the China Center for Type Culture Collection (CCTCC, Wuhan University, Hubei, China). Hela, A549, MCF-7, MDA-MB-231, BEL-7402, MRC-5, and Hep G2 cells were maintained in DMEM media, while HNEpC cells were cultured in RPMI 1640. All media were supplemented with 10% FBS (v/v) and 1% penicillin-streptomycin, and cells were grown in a humidified atmosphere containing 5% CO₂ at 37 °C.

3.6.2. Cell Viability Assay

The MTT assay was conducted to assess cell viability, as previously described [28]. Cells were seeded onto 96-well plates at a density of 1 × 10⁴ cells per well, with each experiment replicated eight times to ensure reproducibility. Following incubation at 37 °C for 24 h, cells were exposed to various concentrations (0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 µM) of compounds 1–10 for 24 h. Subsequently, 10 μL MTT reagent was added to each well, and the plates were incubated for a further 4 h at 37 °C, adhering strictly to the manufacturer’s instructions. The optical density (OD) values were then measured at 570 nm using a microplate reader. The cell survival rate for each compound was calculated employing the following formula:

$$\left({\displaystyle \frac{\mathrm{O}\mathrm{D} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{e}\mathrm{x}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}-\mathrm{O}\mathrm{D} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}}{\mathrm{O}\mathrm{D} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}-\mathrm{O}\mathrm{D} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{B}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}}}\right)\times 100\%$$

3.7. α-Glycosidase Inhibitory Assays

The anti-α-glycosidase effects of compound 1–10 were screened using a previously reported method. Briefly, 40 µL of each target compound 1–10 at various concentrations and 20 µL of α-glycosidase solution (0.25 U/mL) were added to individual wells of a 96-well plate, constituting the experimental group (EG). These were incubated for 10 min at 37 °C. For the sample background control group (SBCG), 40 μL of solutions containing compounds 1–10 and 20 µL of phosphate buffer solution (50 mM) were added. In the blank group (BG), 20 µL of α-glucosidase solution (0.25 U/mL) and 40 µL of 50 mmol/L phosphate buffer solution (PBS) were combined. Additionally, 60 µL of PBS (50 mM) were added to wells designated as the blank control group (BCG). Subsequently, 50 µL of p-nitrophenyl-α-D-glucopyranoside solution (1 mM), serving as the substrate, were added to each well of the EG and incubated for 30 min. After incubation, the reaction was terminated by adding 50 µL of sodium carbonate solution (0.2 mM). The absorbance of each well was measured at 405 nm and the values were recorded as A₁ for EG, A₂ for SBCG, B₁ for BG, and B₂ for BCG.

The inhibition rate for each compound was calculated using the following formula:

$$(1-{\displaystyle \frac{A_1-A_2}{B_1-B_2}})\times 100\%$$

3.8. Molecular Docking Investigation

Autodock Vina (Version 1.1.2), a protein-ligand docking software, was used to evaluate the binding affinity and interaction modes of compounds 1–10 with their target. The 3D coordinates of α-glucosidase (PDB ID: 3A4A, resolution 1.6 Å) were obtained from the Protein Data Bank (RCSB PDB. Available online: https://www.rcsb.org, Accessed on 8 September 2024). All water molecules were removed, and polar hydrogen atoms were added. Grid boxes were designed to encompass the structural domains of each protein, permitting the ligands to move freely. The docking pocket was defined as a cubic region measuring 126 Å × 126 Å × 126 Å, with grid spacing set at 0.375 Å. Optimal molecular interactions were determined by the binding characteristics of α-glucosidase residues and their corresponding binding affinity scores.

3.9. Statistical Analysis

Statistical analysis data from the cytotoxicity and α-glycosidase inhibitory assays were analyzed using the mean and standard deviation. The 50% cytotoxic and inhibitory concentrations were calculated and compared to the control via non-linear regression. These analyses were performed using SPSS^® Statistics (Version 18.0, IBM Software, Armonk, NY, USA).

4. Conclusions

This study examined the bioactive constituents and activities of Camellia flavida as sources for tea and functional foods. Ten new glycoside compounds (flavidosides A–J), comprising three oleanane-type triterpenoid saponins and seven flavonoid glycosides, were isolated from the flowers of C. flavida. Tests were conducted on the cytotoxic effects of the compounds against cancer cells and their α-glucosidase inhibitory activity. The IC₅₀ values recorded for flavidoside C against MCF-7 and BEL-7402 cells were 1.65 ± 0.39 and 4.94 ± 0.41 μM, respectively. The semi-inhibitory concentration of flavidoside H on α-glucosidase was 1.17 ± 0.30 mM. These findings suggest that the bioactive constituents in C. flavida could potentially slow tumor growth and inhibit α-glucosidase activity, highlighting their promise for further research in anti-tumor treatments and metabolic regulation. We are now planning to undertake more comprehensive research to gain a deeper understanding of the underlying mechanisms, with the goal of identifying superior lead compounds.