Nitrogen application influences quality, pharmacological activities and metabolite profiles of Athrixia phylicoides DC. (Bush tea) cultivated under greenhouse and field conditions

Figures & data

Table 1. Weather data of field-cultivated bush tea in Mamelodi, Pretoria, Gauteng Province, South Africa.

Month	Temperature (°C)		Relative humidity (%)		VPD (kPa)	Rainfall (mm)	ET0 (mm)
	Average					Total
	Max	Min	Max	Min
October’14	29.76	11.91	79.03	19.6	1.61	29.97	165.72
November	27.85	14.35	86.35	34.7	1.08	94.74	131.34
December	28.73	16.35	89.48	39.75	0.99	175.26	147.23
January’15	30.18	16.53	89.23	35.29	1.13	136.40	161.30
February	31.85	16.04	88.24	27.01	1.35	32.51	151.64
March	30.18	14.73	88.13	29.29	1.2	71.63	135.49
April	27.42	10.81	90.79	30.57	0.94	43.69	101.49
May	27.56	6.12	83.96	18.55	1.15	0.00	94.86
June	21.26	2.94	84.52	24.92	0.77	1.02	71.00
July	22.27	3.17	83.09	22.7	0.85	0.00	69.11
August	27.26	5.97	76.57	15.15	1.35	0.00	89.53
September	28.10	11.08	79.43	23.39	1.43	56.64	121.77
October	32.58	14.16	77	17.47	1.96	9.40	161.52
November	31.77	13.95	68.24	16.18	2.08	29.72	176.03
December	33.88	18.09	80.00	21.73	1.95	60.20	176.96

*VPD: vapour pressure deficit, ET₀: evapo-transpirative demand, Max: maximum, Min: minimum.

Table 2. Chemical characteristics of soils collected from the field experimental site during the 2014–2015 growing season.

Depth (cm)	P	N	K	Mg	Fe	Ca	pH
	Mg/kg
0–30	52.43	0.01	187.67	275.33	11.46	815.33	7.05
30–60	53.1	0.03	175.33	329.33	8.75	195.00	7.20

Table 3. Response of phytochemical constituents of nitrogen-treated bush tea in a greenhouse environment.

Applied nitrogen (kg/ha)	Leaf tissue nitrogen (%)	Total polyphenols (GAE/g dry matter)	Total flavonoids (QE/g dry matter)	Total tannins (GAE/g dry matter)	Total antioxidant (µg/ml)
0	1.4^d	4.2^e	44.3^c	7.4^d	22.8^c
75	2.4^c	5.6^d	77.6^a	13.1^b	29.4^a
150	3.1^b	6.8^c	78.3^a	15.3^a	29.0^a
225	3.8^a	7.5^b	50.9^b	12.8^b	26.3^b
300	3.7^a	8.4^b	46.5^c	12.1^b	23.1^c
375	3.6^a	9.9^a	45.9^c	9.5^c	16.2^d
Significant response	Q*	L*	Q**	Q*	Q*

Means with the same letters within the column are not significantly different using Duncan Multiple Range Test (DMRT) at 1%.

**Significant quadratic effects (Q) and (L) at 0.01.

GAE: garlic acid equivalent; QE: quercetin equivalent.

Table 4. Response phytochemical constituents of nitrogen-treated bush tea under field conditions.

Applied nitrogen (kg/ha)	Leaf tissue nitrogen (%)	Total polyphenols (GAE/g dry matter)	Total flavonoids (QE/g dry matter)	Total tannins (GAE/g dry matter)	Total antioxidant (µg/ml)
0	1.6^d	7.1^d	16.1^d	8.5^d	52.8^c
75	3.2^c	12.6^c	94.4^a	34.4^a	72.4^a
150	3.5^bc	19.4^a	44.5^b	23.1^b	74.2^a
225	3.8^ab	14.8^b	28.9^c	22.8^b	68.3^b
300	3.7^ab	14.8^b	20.3^c	18.5^c	65.2^b
375	3.6^ab	10.9^c	16.1^d	8.5^d	40.2^d
Significant response	Q*	Q**	Q**	Q*	Q**

Means with the same letters within the column are not significantly different using Duncan Multiple Range Test (DMRT) at 1%.

**Significant quadratic effects (Q) at 0.01.

GAE: garlic acid equivalent and QE: quercetin equivalent.

Figure 1. Least squares regressions leaf nitrogen against leaf total polyphenols concentration of the bush tea leaves harvested from the greenhouse trial.

Figure 2. Least squares regressions leaf nitrogen against leaf total polyphenols concentration of the bush tea leaves harvested from the field trial.

Table 5. Minimum inhibition (MIC) concentration of nitrogen-treated bush tea on selected micro-organisms.

Applied nitrogen (kg/ha)	MIC (mg/ml)
	C. albicans	E. coli	K. oxytoca	K. pneumoniae	P. vulgaris	S. typhi	S. marcescens	S. aureus
0	3.1^b	3.1^c	6.3^a	3.1^a	6.3^a	12.5^a	12.5^a	6.3^a
75	6.3^a	3.1^c	6.3^a	3.1^a	6.3^a	12.5^a	12.5^a	6.3^a
150	6.3^a	3.1^c	6.3^a	3.1^a	6.3^a	12.5^a	12.5^a	6.3^a
225	6.3^a	6.3^b	6.3^a	3.1^a	6.3^a	12.5^a	12.5^a	6.3^a
300	6.3^a	12.5^a	6.3^a	3.1^a	6.3^a	12.5^a	12.5^a	6.3^a
375	6.3^a	12.5^a	6.3^a	3.1^a	6.3^a	12.5^a	12.5^a	6.3^a
Ciprofloxacin	0.196^c	0.196^d	0.196^b	0.196^b	0.196^b	0.196^b	0.196^b	0.196^b

Means with the same letters within the column are not significantly different using Duncan Multiple Range Test (DMRT) at 0.05.

Table 6. Minimum microbicide concentration (MMC) of nitrogen-treated bush tea on selected micro-organisms.

Nitrogen applied (kg/ha)	MIC (mg/ml)
	C. albicans	E. coli	K. oxytoca	K. pneumoniae	P. vulgaris	S. typhi	S. marcescens	S. aureus
0	6.3^b	3.1^b	25.0^a	6.3^b	25.0^a	25.0^a	25.0^a	12.5^a
75	6.3^b	12.5^a	25.0^a	12.5^a	25.0^a	25.0^a	25.0^a	25.0^a
150	6.3^b	12.5^a	25.0^a	12.5^a	25.0^a	25.0^a	25.0^a	25.0^a
225	25.0^a	12.5^a	25.0^a	12.5^a	25.0^a	25.0^a	25.0^a	25.0^a
300	25.0^a	12.5^a	25.0^a	12.5^a	25.0^a	25.0^a	25.0^a	25.0^a
375	25.0^a	12.5^a	25.0^a	12.5^a	25.0^a	25.0^a	25.0^a	25.0^a
Ciprofloxacin	0.196^c	0.196^c	0.196^b	0.196^c	0.196^b	0.196^b	0.196^b	0.196^b

Means with the same letters within the column are not significantly different using Duncan Multiple Range Test (DMRT) at 0.05.

Table 7. Field-harvested bush tea cytotoxicity in relation to nitrogen nutrition.

Nitrogen applied (Kg/ha)	IC50	R^2
Actinomycin D	93.2	0.72
0	125.1	0.94
75	213.7	0.19
150	235.3	0.63
225	219.2	0.76
300	109.7	0.83
375	102.9	0.90

Figure 3. (A) PCA of GC-MS Athrixia phylicoides arboreal samples, (B) Loadings plot of bush tea arboreal samples.

Table 8. Compound detected and concentrations using GC-MS on nitrogen-treated bush tea.

Compound	Retention index	Compounds concentration
		0 kg/ha N	75 kg/ha N	150 kg/ha N	225 kg/ha N	300 kg/ha N	375 kg/ha N
(E)-4,8-dimethyl-1,3,7-nonatriene	1303.00	1.92 ± 0.92	1	1	1	1	1
1,8-cineole	1032.00	1	1	1	1	1	1
1-hexanol,2,2-dimethyl-	975.00	1	1	1	1.51 ± 0.51	1	1
1-Nonene	890.00	1.46 ± 0.46	1 ± 0	1.46 ± 0.46	1.46 ± 0.46	1	1
2-hexenal, (e)-	814.00	1	1	1	1	1.82 ± 0.82	1
3-(1,3-dihydroxyisopropyl)-1,5,8	1156.00	1	1	1	1	2.78 ± 1.78	1
3-chlorodecane	1156.00	1	1.46 ± 0.46	1	1	1	1
3-hexen-1-ol, acetate, (z)-	1009.00	2.91 ± 0.45	1	1	1	2.01	1
Acetic acid	638.00	1	1	1	1	1	1
Alpha-phellandrene	969.00	1.81 ± 0.72	1	1	1.80	1	1
Alpha-pipene	948.00	2.62	2.62	2.62	2.62	2.62	1.54 ± 0.54
Benzene, 1,2,3,5-tetramethyl-	1108.00	1	1.88 ± 0.88	1	1	1	1
Benzene, 1-methyl-4-(1-methylethyl)-	1014.00
Beta phelladrene	1026.00	1	1	1.77 ± 0.77	1	1	1
Beta-myrcene	958.00
Beta-pinene	990.00	2.90 ± 0.01	2.56 ± 0.63	2.25 ± 0.63	2.88	1.63 ± 0.63	1
Beta-cymene	1153.40	1	1	1	1	1.88 ± 0.88	1
Cis-3-hexenyl acetate		1	1	1	1.95 ± 0.95	1	1
Cis-ocimene		1.81 ± 0.81	1	1	1.80 ± 0.80	1	1
Cis-3-hexenol	868.00	1	1	1	2.06 ± 1.06	1	1
Cymene	1187.51	2.76 ± 0.88	1.88 ± 0.88	1	2.76 ± 0.88	1	1
Dichloromethane	937.20	1.45 ± 0.45	1	1	1	1	1
Ethylene oxide cyclic hexamer	2092.00	1	1	4.63 ± 1.81	2.92 ± 1.92	6.63 ± 0.17	1
Ethylene oxide cyclic pentamer	2092.00	1	1	2.89 ± 1.89	2.95 ± 1.95	2.97 ± 1.97	2.82 ± 1.82
Ethylene oxide cyclic tetramer	1395.00	1	6.73 ± 0.12	1	1	2.92 ± 1.92	1
Gamma-terpinene -	1051.00	1	1	1.83 ± 0.83	1	1	1
Heptane, 2,2,4,6,6-pentamethyl-	981.00	1.46 ± 0.46	1	1.93 ± 0.46	1.93 ± 0.46	1	1
Limonene	1020.00	1.77 ± 0.77	3.28	2.51 ± 0.76	1.75 ± 0.75	1	1.75 ± 0.75
Myrcene	995.00	3.16 ± 0.06	3.08	2.38 ± 0.69	2.38 ± 0.69	2.38 ± 0.69	1.69 ± 0.69
Ocimene		1	1.81 ± 0.81	1	1	1	1
O-Cymol		1	1	1.88 ± 0.88	1	1	1
p-Cymene	1012.00	1.89 ± 0.89	1.88 ± 0.88	1.88 ± 0.88	1.88 ± 0.88	1	1
Pentaethylenglycol	1814.00	1	2.84 ± 1.84	1	1	1	1
Pentaoxacyclopentadecane	1804.00	1	1	1	1	1	1
Pyridine	721.70	1	1.79 ± 0.79	1	2.58 ± 0.79	1	1.79 ± 0.79
Sabinene	962.00	2.98 ± 0.05	1	1.64 ± 0.64	2.28 ± 0.64	1	1
Trans-beta-ocimene	976.00	1.81 ± 0.81	1.8 ± 0.80	1	1	1.81 ± 0.81	1

Figure 4. (A) PCA of A. phylicoides arboreal samples run on LC-MS, (B) Loadings plot of the bush tea LC-MS sample.