In vitro inhibition of hyaluronidase by sodium copper chlorophyllin complex and chlorophyllin analogs

Figures & data

Figure 1 Inhibition of hyaluronidase as a function of concentration of test materials.

Figure 2 Structure of copper isochlorin e4.

Notes: Structure provided by: Frontier Scientific, Logan, Utah.

Table 1 Effect of sodium copper chlorophyllin complex, disodium copper isochlorin e4 analogs, and sodium magnesium chlorophyllin complex on hyaluronidase activity in vitro

Test material	Concentration (μg/mL)	Inhibition of hyaluronidase (%)	P-value
Water	–	0	–
Tannic acid	50	100	0.000
Sodium copper chlorophyllin complex	100	100	0.000
	10	13	0.000
	2	1	0.019
	0.4	0	0.135
Copper (II) isochlorin e4 disodium salt	100	100	0.000
	10	24	0.034
	2	0	0.600
	0.4	0	0.839
Oxidized form of copper (II) isochlorin e4 disodium salt	100	83	0.000
	10	2	0.089
	2	0	1.000
	0.4	0	1.000
Chlorophyll MM (sodium magnesium chlorophyllin complex)	100	11	0.000
	10	1	0.839
	2	0	1.000
	0.4	0	1.000

Table 2 Effect of sodium copper chlorophyllin complex alone and in combination with ascorbate analogs on hyaluronidase activity in vitro

Test material	Concentration (μg/mL)	Inhibition of hyaluronidase (%)	P-value
H2O	–	0	1.000
Ethanol, 1%	–	0	0.650
Tannic acid	50	83	0.000
Sodium copper chlorophyllin complex	100	100	0.000
	20	72	0.001
	5	5	0.072
Sodium copper chlorophyllin complex + sodium ascorbate	100	100	0.000
	20	78	0.000
	5	4	0.033
Sodium copper chlorophyllin complex + ascorbyl PM (magnesium ascorbyl phosphate)	100	72	0.000
	20	22	0.000
	5	0	0.919
Sodium copper chlorophyllin complex + ascorbyl palmitate (6-O-palmitoyl-l-ascorbic acid)	100	89	0.000
	20	89	0.000
	5	8	0.000
Sodium copper chlorophyllin complex + dipalmitoyl ascorbate	100	100	0.000
	20	83	0.003
	5	4	0.035

Table 3 Effect of sodium copper chlorophyllin small molecule analogs on hyaluronidase activity in vitro

Test material	Concentration (μg/mL)	Inhibition of hyaluronidase (%)	P-value
H2O	–	0	1
Tannic acid	50	99	0.000
Copper (II) isochlorin e4 disodium salt (MW =658.16)	100	100	0.000
	20	60	0.000
	5	14	0.053
Isochlorin e4 disodium salt (MW =596.63)	100	55	0.000
	20	22	0.001
	5	14	0.017
Copper (II) chlorin e4 disodium salt (MW =658.16)	100	0	0.616
	20	12	0.079
	5	0	0.735
Chlorin e4 disodiumsalt (MW =596.63)	100	17	0.006
	20	5	0.299
	5	9	0.115
Copper (II) chlorin e6 trisodium salt (MW =724.15)	100	24	0.006
	20	0	0.436
	5	5	0.382
Chlorin e6 trisodium salt (MW =662.62)	100	21	0.006
	20	20	0.002
	5	21	0.001

Abbreviation: MW, molecular weight.

Figure 3 HPLC chromatogram of sodium copper chlorophyllin, item number P5521 from Food Ingredient Solutions, lot number 200/98 (“old lot”).

Notes: HPLC analysis was performed using YMC C30 column, (250×4.6 mm), 405 nm. The gradient program used was: 0–50% B over 30 minutes, 50–100% B over 10 minutes, 100% B for 5 minutes and 100–0% B over 5 minutes. Solvent A: methanol:water: acetic acid (90:10:0.5) and Solvent B: tertbutyl methyl ether:methanol:acetic acid (100:10:0.5) at a flow rate of 1.1 mL/min.
Abbreviation: HPLC, high-performance liquid chromatography.

Table 4 Analysis of old and new lots of commercial sodium copper chlorophyllin samples for copper isochlorin e4 and oxidized copper isochlorin e4

Component	Food solutions		Spectrum
	Old lot	New lot	Old lot	New lot
Copper isochlorin e4, disodium salt	41%	24%	None detected	50%
Oxidized copper isochlorin e4, disodium salt	26%	18.7%	21%	12%